Stratification of acute myeloid leukemia patients for sensitivity to kinase pathway inhibitor therapy

Abstract

The present invention relates to the treatment of acute myeloid leukemia (AML) in patients. Specifically, the present invention relates to improved methods for identifying AML patients that can be effectively treated with kinase pathway inhibitors and improved methods for predicting whether kinase pathway inhibitors will be effective in treating AML in an individual patient. The present invention also includes methods for screening multiple patients suffering from acute myeloid leukemia to determine whether the acute myeloid leukemia of any one or more of these patients can be effectively treated with a kinase pathway inhibitor. The present invention further provides methods of treating such patients with kinase pathway inhibitors and kinase pathway inhibitors for use in such methods.

Description

[Technical field]

[0001] The present invention relates to the treatment of acute myeloid leukemia (AML) in patients. In particular, the present invention relates to improved methods for identifying AML patients that may be effectively treated with kinase pathway inhibitors, and improved methods for predicting whether a kinase pathway inhibitor may be effective in treating AML in an individual patient. The present invention also includes a method for screening patients suffering from acute myeloid leukemia to determine whether the acute myeloid leukemia of any one or more of these patients may be effectively treated with a kinase pathway inhibitor. The present invention further provides methods of treatment of such patients with kinase pathway inhibitors, and kinase pathway inhibitors for use in such methods. [Background technology]

[0002] Acute myeloid leukemia - Disease characteristics and classification Acute myeloid leukaemia (AML), also known as acute myeloblastic leukemia, acute granulocytic leukemia, or acute nonlymphoblastic leukemia, is an aggressive cancer of the blood and bone marrow. AML is characterized by the excessive production of immature white blood cells (known as myeloblasts) by the bone marrow. In healthy individuals, the blasts normally develop into mature white blood cells. However, in AML, the blasts do not differentiate normally and remain in an early quiescent state of development.

[0003] In AML, the bone marrow may also produce abnormal red blood cells and platelets. The number of these abnormal cells increases rapidly and begins to crowd out the normal white blood cells, red blood cells, and platelets that the body needs. If left untreated, acute myeloid leukemia is rapidly fatal.

[0004] Various classification systems have been devised to classify AML into disease subtypes, with the aim of allowing a more accurate prognosis of disease progression and identifying the most appropriate form of treatment. The earliest system was the French-American-British (FAB) classification, first devised in the 1970s by a group of leukemia experts from France, the United States, and the United Kingdom. This system divides AML into subtypes according to the type of cell from which the leukemia originates and the stage of maturation that the myeloblastic cells have reached at the time of quiescence. Subtypes M0 to M5 originate from white blood cell precursors and range from anaplastic myeloblastic leukemia (M0) to monocytic leukemia (M5). Subtype M6 originates from a very early form of red blood cells (erythrocytic leukemia), and subtype M7 AML begins with an early form of cells that form platelets (megakaryoblastic leukemia).

[0005] Under the FAB system, AML is classified by visual inspection of cytomorphological features under a microscope and identification of various chromosomal abnormalities. An updated version of the FAB classification was published in 1985 - see Bennett et al., Proposed revised criteria for the classification of acute myeloid leukaemia, Ann Intern Med 1985;103(4):620-625.

[0006] The level of knowledge in this field has advanced considerably since the FAB system was first devised in the 1970s. Although the system has been updated to incorporate some of this knowledge, it was felt necessary to create a new classification system, taking into account additional factors now known to influence prognosis and be crucial to optimizing effective treatment.

[0007] Thus, the World Health Organization (WHO) classification system divides AML into several broad groups. These include: AML with recurrent genetic abnormalities (specific chromosomal changes) AML with multilineage dysplasia Related to previous cell-damaging treatments (e.g. chemotherapy and radiation therapy) and AML, also known as therapy-related myeloid neoplasms. AML not otherwise classified - e.g. · Undifferentiated AML (M0) AML with minimal maturity (M1) AML with maturation (M2) Acute myelomonocytic leukemia (M4) Acute monocytic leukemia (M5) · Acute red blood cell leukemia (M6) Acute megakaryoblastic leukemia (M7) Acute basophilic leukemia Acute panmyelopathy with fibrosis · Myeloid sarcoma (also known as granulocytic sarcoma or chloroma).

[0008] In addition to these two major classification systems, AML is further classified and subtyped by reference to certain molecular markers that have been shown to correlate with specific phenotypes and outcomes. For example, patients with mutations in the NPM1 and CEBPA genes are known to have better long-term outcomes, and patients with certain mutations in FLT3 have been shown to have a poorer prognosis - see Yohe et al., J Clin Med. 2015 Mar 4(3):460-478.

[0009] Current treatment regime Conventional treatments for AML include chemotherapy and radiation therapy as well as stem cell and bone marrow transplants. Most patients respond well to such treatments initially, but relapse rates are high and patients generally become resistant to first-line treatment. Overall, the five-year survival rate for AML patients undergoing conventional treatment is approximately 26%.

[0010] Historically, except for acute promyelocytic leukemia, the treatment of AML has not targeted disease subtypes. Rather, the classification of AML according to the above-mentioned system has primarily served to inform clinical decisions regarding the appropriate intensity of treatment. However, in recent years, efforts have been made to identify targeted forms of treatment suitable for specific disease types and patient subgroups. Kinase pathway inhibitors have been of particular interest as a potential novel personalized treatment for AML. Recently, the FLT3 inhibitor midostaurin has been approved by the FDA for the treatment of adult patients with newly diagnosed AML who have specific activating mutations in the FLT3 gene. The preclinical efficacy of MEK inhibitors for the treatment of AML with oncogenic NRAS mutations has also been investigated (Burgess et al., Blood 2014;124(26):3947-3955). However, kinase pathway inhibitors are not yet routinely used to treat AML, and the sensitivity of these screening protocols to reliably identify patients who will be sensitive to kinase pathway inhibitor treatment has not been fully tested. Summary of the Invention [Problem to be solved by the invention]

[0011] Thus, there remains a need for improved methods for targeted treatment of AML, and in particular, there remains a need for improved methods for identifying patients who will respond to treatment with kinase pathway inhibitors and those who will not respond to such treatment. Accurate and sensitive stratification is necessary not only to ensure that patients who will respond to a particular treatment can be identified as such and appropriately treated, but also to ensure that non-responding patients are not treated unnecessarily. [Means for solving the problem]

[0012] The present inventors have integrated proteomic, kinomic and genomic profiling to investigate the mechanism that sensitizes primary AML cells to kinase pathway inhibitors, thereby enabling the identification of a panel of biomarkers that accurately identifies sensitive cells.The present inventors have discovered that more differentiated leukemia cells show higher sensitivity to kinase pathway inhibitors than less differentiated leukemia cells. Thus, the differentiation state of leukemic cells, which can be assessed according to the protocols and criteria described herein, provides an effective biomarker for identifying leukemic cells and patients that are sensitive to kinase pathway inhibitors, allowing novel and effective stratification of patients for kinase inhibitor therapy. The inventors further identified a correlation between specific genetic mutations and kinase pathway inhibitor sensitivity, which, among other things, has the potential to provide an effective companion diagnostic test for use in combination with kinase inhibitor therapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Thus, in a first aspect, the present invention provides a method of predicting the efficacy of a kinase pathway inhibitor in the treatment of acute myeloid leukemia in an individual patient, the kinase pathway inhibitor inhibiting a kinase signaling pathway involved in cell proliferation or cell survival, the method comprising: (a) determining the differentiation status of the patient's leukemia; and (b) predicting that the patient's acute myeloid leukemia may be effectively treated with the kinase pathway inhibitor if the differentiation state of the leukemia is advanced. The present invention provides a method comprising:

[0014] In a second aspect, the present invention provides a method of treating acute myeloid leukemia in an individual patient, comprising: (a) determining the differentiation status of the patient's leukemia; and (b) if the differentiation state of the leukemia is advanced, treating the patient with a kinase pathway inhibitor that inhibits a kinase signaling pathway involved in cell proliferation or cell survival; The present invention provides a method comprising:

[0015] In a third aspect, the invention provides a method of screening a plurality of patients having acute myeloid leukemia to determine whether acute myeloid leukemia in any one or more of said plurality of patients can be effectively treated with a kinase pathway inhibitor, which inhibits a kinase signaling pathway involved in cell proliferation or cell survival, the method comprising: (a) determining, for each patient, the differentiation status of the patient's leukemia; and (b) identifying any one or more patients having an advanced differentiation state leukemia as having a leukemia suitable for effective treatment with said kinase pathway inhibitor; The present invention provides a method comprising:

[0016] In a fourth aspect, the present invention provides a kinase pathway inhibitor for use in a method of treating acute myeloid leukemia in an individual patient, the kinase pathway inhibitor inhibiting a kinase pathway involved in cell proliferation or cell survival, the treatment comprising: (a) determining the differentiation status of this patient's leukemia; and (b) if the leukemic cells are undergoing differentiation, treating the patient with said kinase pathway inhibitor; The present invention provides a kinase pathway inhibitor comprising:

[0017] The present invention further provides a computational implementation of the method of screening according to the third aspect of the invention and the method of predicting the efficacy of kinase pathway inhibitor therapy according to the first aspect of the invention. The present invention also provides software for performing either or both of these computer-implemented methods.

[0018] Each of these aspects of the invention comprises a step (a) of determining the differentiation state of the patient's leukemia. As explained above, AML involves the proliferation of abnormal, partially differentiated myeloblasts. The term "the differentiation state of the patient's leukemia" therefore refers to the differentiation state of the patient's leukemia cells. Thus, suitably, step (a) may comprise determining the differentiation state of leukemia cells already obtained from the patient. Alternatively, step (a) may further comprise obtaining leukemia cells from the patient before determining the differentiation state of said leukemia cells. The leukemia cells may be obtained, for example, from a peripheral blood sample or a bone marrow sample. The present invention is applicable to all AML patients (e.g., newly diagnosed (untreated) AML patients, AML patients who have undergone or are undergoing other forms of treatment, and relapsed AML patients).

[0019] Suitably, the differentiation state of a leukemic cell may be determined by analysing data relating to the leukemic cell, as described below. In some embodiments, the data has already been collected and recorded, and step (a) comprises obtaining or receiving the data for analysis. In other embodiments, step (a) further comprises collecting and recording the data for analysis, as described below. In some embodiments, the step of determining the differentiation state of a patient's leukemic may consist of determining whether the patient's leukemic is progressing (a binary (yes / no) decision).

[0020] The differentiation state of a patient's leukemia may be determined by analyzing data relating to the morphological and / or cytochemical characteristics of the leukemic cells, and / or by analyzing data relating to the expression, activation, and / or phosphorylation in the leukemic cells of one or more differentiation markers, such as cell surface differentiation markers, and / or functional differentiation markers, such as kinase pathway activity markers, and / or by reference to data recording the classification of the leukemic cells under the French-American-British (FAB) classification as described in Bennett et al., Proposed revised criteria for the classification of acute myeloid leukaemia, Ann Intern Med 1985;103(4):620-625. This data can include any type of information about the cell (for example, but not limited to, information about the cell's appearance, properties, characteristics, genotype, phenotype, activity, classification, and function, as well as for example, but not limited to, images of the cell, written descriptions of the cell, and measurements of any type obtained from the cell).

[0021] The data regarding morphological characteristics of the leukemic cells may include data documenting the appearance of the cells under a light microscope, optionally using a stain such as Romanowsky stain. The data may include, for example, a visual image of the cells and / or a written description of the cells. Step (a) may include analyzing the data to determine whether the cells meet the FAB criteria for identifying M4 cells as defined in Bennett et al., supra. Specifically, the differentiation state may be determined to be advanced if the data indicate that at least 20% of the leukemic cells have the appearance characteristic of granulocytic-monocytic cells, and / or if the data indicate that, among the leukemic cells, myeloblasts, monoblasts, and promonocytes make up 20% or more of the non-erythroid cells, and myeloblasts and granulocytes make up 80% or less of the non-erythroid cells. For example, the differentiation state may be determined to be advanced if the data indicate that at least 20% of the cells have a slightly granular, grayish cytoplasm and folded nuclei characteristic of granulocytic-monocytic cells (M4 FAB). In samples obtained from bone marrow, blast cells (myeloblasts, promyelocytes, myelocytes, and post-granular) constitute more than 30% and less than 80% of the non-erythroid cells, and preferably, but not essentially, in samples obtained from peripheral blood, monocyte counts (monoblasts, promonocytes, and monocytes) are greater than 5×10 9 If the number of cells / L or more, then a differentiated state can be determined to be advanced. See Bennett et al., supra, p. 622.

[0022] In some embodiments, the data regarding morphological characteristics of leukemic cells has already been recorded, and step (a) comprises obtaining the data for analysis. In other embodiments, step (a) further comprises collecting and recording the data regarding morphological characteristics of leukemic cells for analysis. Methods for collecting and recording the data regarding morphological characteristics of cells are conventional and known in the art, and are described in Bennett et al., supra, and others.

[0023] The data regarding the cytochemical characteristics of the leukemic cells may include data documenting the response of the cells to reagents (e.g., Sudan Black B, and / or peroxidase, and / or specific or non-specific esterases). The data may include, for example, visual images of the cells, written descriptions of the cells, flow cytometry data, and other types of cytochemical data. Step (a) may include analyzing the data to determine whether the cells meet the FAB criteria for identifying M4 cells as defined in Bennett et al., supra. Specifically, if the data indicates that at least 20% of the cells are reactive to Sudan Black B, and / or peroxidase, and / or specific or non-specific esterase, the differentiation state may be determined to be advanced (M4 FAB).

[0024] In some embodiments, said data regarding the cytochemical characteristics of white blood cells has already been recorded and step (a) comprises obtaining said data for analysis. In another embodiment, step (a) further comprises collecting and recording said data regarding the cytochemical characteristics of the leukemic cells for analysis. Methods for collecting and recording data regarding the cytochemical characteristics of the cells are conventional and known in the art, and are described in Bennett et al., supra, and others.

[0025] The data regarding the expression, activation, and / or phosphorylation of one or more differentiation markers and / or functional differentiation markers, such as cell surface differentiation markers, in leukemic cells may include data documenting the presence or absence or expression level of one or more cell surface differentiation markers, such as signaling molecules, on the surface of the leukemic cells, which cell surface differentiation markers are generally expressed or overexpressed on healthy myelomonocytic cells and which cell surface differentiation markers are generally not expressed or overexpressed on undifferentiated myeloblasts, wherein the presence of the one or more cell surface differentiation markers on the leukemic cells or the expression of the one or more cell surface differentiation markers at high levels on the leukemic cells indicates an advanced differentiation state. The data may include, for example, a written description of the cells, or any type of data obtained from an assay measuring cell surface protein expression (e.g., mass cytometry, or any other technique or assay known in the art). In some embodiments, the data is already recorded, and step (a) involves obtaining the data for analysis. In other embodiments, step (a) further involves collecting and recording the data for analysis according to standard conventional methods and protocols known in the art (e.g., by mass cytometry).

[0026] The cell surface differentiation markers may comprise a panel of cell surface marker proteins including one or more of CD3, CD7, CD11b, CD11c (integrin alpha-X, ITAX), CD14, CD15, CD16, CD18 (integrin beta, ITB2), CD19, CD33, CD34, CD35 (CR1), CD38, CD44, CD45, CD64, CD97, CD117, CD123, CD180, CD184, HLA-C (1C02), APOBR, platelet membrane receptor Gi24 (VSIR), and HLA-DR; and / or any cell surface protein expressed in conjunction with said one or more cell surface marker proteins. CD markers (also known as cluster of differentiation markers) are well-defined subsets of cell surface receptors (epitopes) that are specific for cell types and differentiation stages and are recognized by antibodies. All of the cell surface marker proteins listed above are known in the art and have been well characterized - see, e.g., Zola et al., H, (2007) "CD molecules 2006-human cell differentiation molecules." J Immunol Methods. 319(1-2):1-5. These cell surface marker proteins have been shown to be generally expressed at high levels on the surface of leukemic cells with an advanced differentiation state that is sensitive to kinase pathway inhibitors, but generally not expressed on undifferentiated myeloblasts. Thus, step (a) may include analysing the data to determine whether a panel of cell surface marker proteins is expressed or expressed at high levels by the white blood cells, and determining that the differentiation state is progressing if the panel of cell surface marker proteins is expressed or expressed at high levels. Preferably, the panel of cell surface marker proteins includes any two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen, or fifteen, or sixteen, or seventeen, or eighteen, or all of CD11b, CD11c (integrin alpha-X, ITAX), CD14, CD15, CD16, CD18 (integrin beta, ITB2), CD33, CD35 (CR1), CD38, CD44, CD45, CD64, CD97, CD123, CD180, HLA-C (1C02), APOBR, platelet membrane receptor Gi24 (VSIR), and HLA-DR.

[0027] Reference to expression of one or more cell surface proteins (e.g., cell surface marker proteins) at a "high level," as used here and elsewhere herein, indicates a level of expression that is higher compared to the average level of expression of the relevant cell surface protein. Reference to a "low level" of expression similarly indicates a level of expression that is below the average level of expression of a cell surface protein. The average level of expression of a cell surface protein is a standardized value, which may be determined by reference to the average calculated across multiple samples, or may be determined by reference to the level of expression of the cell surface protein in undifferentiated myeloblasts or other healthy cell types, which may be established by laboratory analysis according to methods well known in the art (e.g., LC-MS / MS) or by reference to information available in the art. Thus, for example, the average level of expression of a cell surface protein may be determined by establishing the range of expression levels of the cell surface protein in cell samples obtained from a large number of AML patients and calculating the average level of expression across these samples. A "high level" of expression of a cell surface protein refers to a level of expression that is higher than this calculated average.

[0028] Optionally, the panel of cell surface marker proteins may further include one or more of CD19, CD117, CD7, CD34, CD3, and CD184. The panel of cell surface marker proteins may advantageously comprise CD45, and / or CD11b, and / or CD44, and / or CD14, and / or CD16, and / or CD64, and / or CD15. Specifically, the panel of cell surface marker proteins may include any one of CD45, CD11b, CD44, CD14, CD16, CD64, and CD15, or may include any two, three, four, five, or six of CD45, CD11b, CD44, CD14, CD16, CD64, and CD15. Suitably, the panel of cell surface marker proteins may consist of CD45, CD11b, CD44, CD14, CD16, CD64, and CD15. In some preferred embodiments, the panel of cell surface markers consists of any one, two, three, four, five, six, seven, eight, nine, ten, or all of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR. In other preferred embodiments, the panel of cell surface marker proteins consists of any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all of: CD3, CD7, CD11b, CD14, CD15, CD16, CD19, CD33, CD34, CD38, CD44, CD45, CD64, CD117, CD123, CD184, and HLA-DR.

[0029] The data regarding expression, activation, and / or phosphorylation in leukemic cells of one or more differentiation markers, such as cell surface differentiation markers and / or functional differentiation markers, may additionally or alternatively include data recording the expression, activation, and / or phosphorylation of one or more functional differentiation markers, which are generally expressed, overexpressed, activated, and / or phosphorylated in healthy monomyeloelocytic cells and which are generally not expressed, overexpressed, activated, and / or phosphorylated in undifferentiated myeloblasts, and wherein the expression, activation, and / or phosphorylation of the one or more functional differentiation markers indicates an advanced differentiation state. The data may include, for example, a written description of the cells, or any type of data obtained from assays measuring the expression, activation, or phosphorylation of cellular proteins according to any technique known in the art (e.g., LC-MS / MS analysis, or immunochemical techniques such as Western blotting, ELISA, and reverse phase protein assays). In some embodiments, the data has already been recorded and step (a) comprises obtaining the data for analysis. In other embodiments, step (a) further comprises collecting and recording said data for analysis according to standard conventional methods known in the art (eg, by LC-MS / MS).

[0030] The one or more functional differentiation markers may comprise a panel of protein markers including one or more enzymes, integrins, kinases, phosphatases, signal transduction regulators, cytoplasmic proteins and phosphoproteins, membrane proteins and phosphoproteins, such as GTPases or cytoplasmic phosphoproteins involved in other forms of cell signaling, which protein markers are generally expressed, overexpressed, and / or activated in healthy myelomonocytic cells and generally not expressed, overexpressed, and / or activated in undifferentiated myeloblasts.

[0031] A panel of protein markers may include any one, two, three, four, five, six, seven, eight, nine, ten, or more of the following: Lysozyme C (LYZ), neutrophil cytosolic factor 2 (NCF2), myeloid cell nuclear differentiation antigen (MNDA), AK1C4, ERG, nesprin-3, voltage-gated hydrogen channel 1, fructose-1,6-bisphosphatase 1, monocyte differentiation antigen CD14, thymidine phosphorylase, CD180 antigen, putative annexin A2-like protein, retinoid-inducible serine carboxypeptidase, annexin A2, Golgi-associated plant pathogenesis-related protein 1, integrin beta-2, BTB / POZ domain-containing protein KCTD12, cytoskeleton-associated protein 4, integrin alpha-X, type 1 complement receptor, annexin A5, uncharacterized protein FLJ45252, galectin-3, adenylate kinase isoenzyme 1 1), protein S100-A10, thiamine-triphosphatase, deoxynucleoside triphosphate triphosphohydrolase SAMHD1, mitochondrial amidoxime reducing component 1, coronin-1B, Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1, granulin, ribonuclease inhibitor, long-chain fatty acid CoA ligase 1, protein S100-A11, procathepsin H, cathepsin S, galectin-1, Transport and Golgi organization protein 2 homolog, Arf-GAP domain and FG repeat-containing protein 1, long-chain fatty acid CoA ligase 3, RasGTPase-activating-like protein IQGAP1, allograft inflammatory factor 1, transcriptional intermediary factor 1-beta, beta-arrestin-2, dihydropyrimidine dehydrogenase [NADP(+)], alpha-N-acetylgalactosaminidase, cathepsin B, aminopeptidase B, lysosomal protective protein, phosphoglycerate mutase 1, polypeptide N-acetylgalactosaminyltransferase 2, cytokine receptor-like factor 3, calpastatin, EF-hand domain-containing protein D2, dual specificity mitogen-activated protein kinase kinase 1 1), major vault protein, alpha-galactosidase A, tyrosine protein kinase SYK, sister chromatid cohesion protein PDS5 homolog B, calpain-2 catalytic subunit, FK506-binding protein 15, protein disulfide isomerase, tensin-3, apolipoprotein B receptor, transforming protein RhoA, plastin-2, actin-associated protein 2 / 3 complex subunit 2, CD97 antigen, cathepsin Z, neuroblast differentiation-associated protein AHNAK, nonconventional myosin-If, and pyruvate acid kinase PKM, protein THEMIS2, plastin-3, type 6 tyrosine-protein phosphatase non-receptor, ezrin, leucine-rich repeat-containing protein 59, guanine nucleotide-binding protein G(I) / G(S) / G(T) subunit beta-2, coronin-1A, radixin, transketolase, growth factor receptor-binding protein 2, type V protein ATPase subunit B (kidney isoform), coatomer subunit epsilon, alpha-soluble NSF attachment protein, Rho GDP-dissociation inhibitor 2, and / or guanine nucleotide-binding protein subunit beta-4, and / or any proteins selectively expressed and / or activated therewith. These protein markers have been found to be generally expressed and / or activated in leukemic cells with an advanced differentiation state that is sensitive to kinase pathway inhibitors, but generally not expressed and / or activated in undifferentiated myeloblasts. Thus, step (a) may comprise analysing the data to determine whether the panel of protein markers is expressed and / or activated in said leukemic cells, and if the panel of protein markers is expressed and / or activated in the cells, determining an advanced differentiation state.

[0032] Advantageously, the panel of protein markers may comprise any one, two, three, four or five of lysozyme C (LYZ), neutrophil cytosolic factor 2 (NCF2), myeloid nuclear differentiation antigen (MNDA), AK1C4 and ERG, and step (a) may comprise analysing the data to determine whether the panel of protein markers is expressed in the leukemic cell, and determining an advanced differentiation state if the panel of protein markers is expressed in the cell.

[0033] The one or more functional differentiation markers may additionally or alternatively comprise a panel of kinase pathway activity markers including one or more kinases, phosphatases, phospholipases, integrins, signal transduction regulators, G proteins, transmembrane receptors, phosphopeptides, and / or other kinase signaling molecules that are typically expressed and / or activated and / or phosphorylated in kinase signaling pathways in healthy myelomonocytic cells, but not typically expressed and / or activated and / or phosphorylated in undifferentiated myeloblasts. Advantageously, the kinase signaling pathway may be one that is inhibited by a kinase pathway inhibitor, so for example, if the kinase pathway inhibitor is a RAS-RAF-MEK-ERK pathway inhibitor such as trametinib, the panel of kinase pathway activity markers may include markers of the RAS-RAF-MEK-ERK signaling pathway.

[0034] In particular, a panel of kinase pathway activity markers may include one or more kinases, phosphatases, phospholipases, integrins, signal transduction regulators, G proteins, transmembrane receptors, and / or other kinase signaling molecules expressed and / or activated in kinase signaling pathways involved in cell proliferation or cell survival. In some embodiments, one or more of the kinases, phosphatases, phospholipases, integrins, signal transduction regulators, G proteins, transmembrane receptors, and / or other kinase signaling molecules may be expressed and / or activated in a kinase signaling pathway that is inhibited by the kinase pathway inhibitor. In some embodiments, one or more of the kinases, phosphatases, phospholipases, integrins, signal transduction regulators, G proteins, transmembrane receptors, and / or other kinase signaling molecules may be inhibited by the kinase pathway inhibitor.

[0035] The panel of kinase pathway activity markers includes FES, PKC and protein kinase C isoforms such as PKC delta (KPCD), PRKCA, PRKCB, and PRKCD, PKA, PAK, such as PAK1 and PAK2, STK10, GSK3A, RSK2, RAS, RAF, MEK, such as MEK1 (MAP2K1), ERK, such as MAPK3 (ERK1) and MAPK1 (ERK2), PI3K, AKT, such as AKT1, MTOR, S6 kinase, STAT5, CAMKK, SYK (KSYK), LYN, P38A, CDK1, CK2A1, PKACA, IRAK4, PKCB The kinases may include any one, two, three, four, five, six, seven, eight, nine, ten, or more of iso2, Cot, PKCD, PKCA, PKCB, PKCG, PKCH, BRAF, MEK2, PDK1, CDK2, PTN6, D3(PLD3), IQGAP1, GRB2, RHOA, RHOG, and S10AB, as well as any kinases, phosphatases, phospholipases, integrins, signal transduction regulators, G proteins, transmembrane receptors, and / or other kinase signaling molecules that are selectively expressed or activated therewith. These kinase pathway activity markers have been found to be generally expressed and / or activated and / or phosphorylated in leukemic cells with an advanced differentiation state that is sensitive to kinase pathway inhibitors, but generally not expressed and / or activated and / or phosphorylated in undifferentiated myeloblasts. Thus, step (a) may comprise analysing the data to determine whether the panel of kinase pathway activity markers is expressed and / or activated and / or phosphorylated in the leukemic cell, and determining an advanced differentiation state if the panel of kinase pathway activity markers is expressed and / or activated and / or phosphorylated in the cell.

[0036] Advantageously, the panel of kinase pathway activity markers may comprise any one, two, three or four of PKC, ERK, PAK1 and P38α, and step (a) may comprise analysing the data to determine whether the panel of kinase pathway activity markers is expressed and activated in the leukemic cell, and determining an advanced differentiation state if the panel of kinase pathway activity markers is expressed and activated in the cell.

[0037] Suitably, the panel of kinase pathway activity markers may comprise any one, two, three, four or five of PKCD, PKCA, PKACA, IRAK4 and CK2A1, and step (a) may comprise analysing the data to determine whether the panel of kinase pathway activity markers is expressed and activated in the leukemic cell, and determining an advanced differentiation state if the panel of kinase pathway activity markers is expressed and activated in the cell. Alternatively, the panel of kinase pathway activity markers may include any one, two, three, four, five, or six of MAPK1, MAPK2, AKT, AKT1S1, MAP2K1, and MAP2K2, and step (a) may include analysing the data to determine whether the panel of kinase pathway activity markers is expressed and activated in the leukemic cell, and determining an advanced differentiation state if the panel of kinase pathway activity markers is expressed and activated in the cell.

[0038] The panel of kinase pathway activity markers may additionally or alternatively include a panel of one or more phosphorylation sites that are generally phosphorylated or generally phosphorylated at high levels in kinase signaling pathways in healthy myelomonocytic cells, but are generally not phosphorylated or generally not phosphorylated at high levels in undifferentiated myeloblasts. Step (a) may comprise analysing the data to determine whether the panel of phosphorylation sites is highly phosphorylated in said leukemic cells, where if the panel of phosphorylation sites is highly phosphorylated in the leukemic cells, then an advanced differentiation state is determined.

[0039] Reference to phosphorylation at a "high level," as used here and elsewhere herein, indicates a level of phosphorylation that is high relative to the average phosphorylation of a reference protein or at a reference phosphorylation site. Reference to a "low level" of phosphorylation similarly indicates a level of phosphorylation that is below the average phosphorylation of a reference protein or at a reference phosphorylation site. The average phosphorylation of a reference protein or reference phosphorylation site is a normalized value, which may be determined by reference to an average calculated across multiple samples, or by reference to the phosphorylation state of the reference protein or reference phosphorylation site in undifferentiated myeloblasts or other healthy cell types, which may be established by laboratory analysis according to methods well known in the art (e.g., LC-MS / MS) or by reference to information available in the art. Thus, for example, the average level of phosphorylation at a particular phosphorylation site can be determined by establishing the range of phosphorylation at this site in cell samples obtained from a number of AML patients and calculating the average phosphorylation across these samples. A "high level" of phosphorylation at this site is a level of phosphorylation that is higher than this calculated average.

[0040] In particular, the panel of phosphorylation sites may include one or more phosphorylation sites that are highly phosphorylated in a kinase signaling pathway involved in cell proliferation or cell survival. In some embodiments, one or more of said phosphorylation sites may be highly phosphorylated in a kinase signaling pathway that is inhibited by said kinase pathway inhibitor.

[0041] The panel of phosphorylation sites may include any one, two, three, four, five, six, seven, eight, nine, ten, or more than ten of the phosphorylation sites listed in Table 1 below. [Table 1] JPEG0007603294000002.jpg249170JPEG0007603294000003.jpg240170JPEG0007603294000004.jpg243170JPEG0007603294000005.jpg246170JPEG0007603294000006.jpg237170JPEG0007603294000007.jpg250170JPEG0007603294000008.jpg243170JPEG0007603294000009.jpg245170JPEG0007603294000010.jpg244170JPEG0007603294000011.jpg247170JPEG0007603294000012.jpg251170JPEG0007603294000013.jpg238170JPEG0007603294000014.jpg242170JPEG0007603294000015.jpg252170JPEG0007603294000016.jpg247170JPEG0007603294000017.jpg249170JPEG0007603294000018.jpg247170JPEG0007603294000019.jpg242170JPEG0007603294000020.jpg243170JPEG0007603294000021.jpg246170JPEG0007603294000022.jpg244170JPEG0007603294000023.jpg248170JPEG0007603294000024.jpg246170JPEG0007603294000025.jpg250170JPEG0007603294000026.jpg242170JPEG0007603294000027.jpg246170JPEG0007603294000028.jpg242170JPEG0007603294000029.jpg238170JPEG0007603294000030.jpg248170JPEG0007603294000031.jpg243170JPEG0007603294000032.jpg252170JPEG0007603294000033.jpg247170JPEG0007603294000034.jpg241170JPEG0007603294000035.jpg243170JPEG0007603294000036.jpg243170JPEG0007603294000037.jpg250170JPEG0007603294000038.jp g241170JPEG0007603294000039.jpg245170JPEG0007603294000040.jpg242170JPEG0007603294000041.jpg244170JPEG0007603294000042.jpg2 46170JPEG0007603294000043.jpg243170JPEG0007603294000044.jpg248170JPEG0007603294000045.jpg240170JPEG0007603294000046.jpg249 170JPEG0007603294000047.jpg241170JPEG0007603294000048.jpg252170JPEG0007603294000049.jpg252170JPEG0007603294000050.jpg76170.

[0042] The panel of phosphorylation sites may include any one, two, three, four, five, six, seven, eight, nine, ten, or more than ten of the phosphorylation sites listed in Table 2 below. [Table 2] JPEG0007603294000052.jpg255170JPEG0007603294000053.jpg255169JPEG00076032940 00054.jpg255169JPEG0007603294000055.jpg254170JPEG0007603294000056.jpg179170

[0043] Advantageously, the panel of phosphorylation sites may comprise one or more phosphorylation sites in a kinase, e.g. PAK1 in S144, PAK2 in S141, MAPK1 at Y187, MAPK1 in T185, RPS6KA1 on S380, MAPK3 in T202, MAPK3 in Y204, MAP3K3 in S166, SYK in S295 and S297, IRAK3 on S110, PKN1(379-396+phosphoST), STK10 (447-464+phosphoST), RIPK3 in S410, PRKCD in T218, PRKCD on T295, PRKCD in Y313, PRKCD in T507, PRKCD in T645, PRKCD in S664, PRKCD in T2638, MARK2 in S535, MAP3K2 in S535, PRKD2 (710-730+phospho-Y), NRK on s805, PRKAR2A, ZAK in S58 (591-616+phospho-ST), MAP4K4 on S900, CDK9 in S347, RPS6KA4 (681-699+2 phospho-ST), MAST3 (1254-1274+phosphoST), NEK9 (10–39+phospho-ST), GSK3A (19-50+ phospho-ST), RPS6KA3 in S369, RIPK2 in S531, AAK1 in T606, TYK2 in Y292, PDPK2 in S214, PRKAA1(3-8+phosphoST), STK11P in S772, BAZ1B in S1468, CLK1 on S140, MAP4K2 in S328, WNK1 (1996~2021+phospho-ST), CDK11A in S271, FES in Y713 and / or may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the TNIKs in S769; Step (a) may include analyzing the data to determine whether the panel of phosphopeptides is phosphorylated at a high level in the leukemic cells, where if the panel of phosphopeptides is phosphorylated at a high level in the cells, then determining an advanced differentiation state.

[0044] Preferably, the panel of phosphorylation sites may include regulatory phosphorylation sites in the kinase, for example PAK1 at S144, PAK2 at S141, MAPK1 at Y187 and / or T185, and RPS6KA1 at S380; Step (a) may include analyzing the data to determine whether a panel of phosphopeptides is highly phosphorylated in the leukemic cells, and determining that if the panel of phosphopeptides is highly phosphorylated in the cells, then the cells are in an advanced differentiation state.

[0045] Suitably, the panel of phosphorylation sites may include MAPK1 at Y187, PAK2 at S141, and PRKCD at Y313, and step (a) may comprise analysing the data to determine whether the panel of phosphopeptides is phosphorylated in the leukemic cells, and determining that if the panel of phosphopeptides is phosphorylated in the cells, then the cells are in an advanced differentiation state.

[0046] Optionally, the panel of phosphorylation sites may include FES at Y713, MAPK3 at T202 / Y204, MAPK1 at T185 / Y187, PAK1 at S144, MEK1 at S222, PAK2 at S141, and PRKCD at S645, and step (a) may include analyzing the data to determine whether the panel of phosphopeptides is phosphorylated in the leukemic cells, and determining that the panel of phosphopeptides is phosphorylated in the leukemic cells is in an advanced differentiation state if the panel of phosphopeptides is phosphorylated in the cells.

[0047] A panel of phosphorylation sites may include, for example, one or more phosphorylation sites on one or more PKC isoforms such as PRKCA, PRKCB, and / or PRKCD, as shown in Table 1, and / or one or more phosphorylation sites on one or more of STK10, GSK3A, PAK1, PAK2, and Gi24 (VSIR). For example, the panel of phosphorylation sites may include S21 of GSK3A, and / or T507, T295, T218, Y313, T507, and / or S664 of PRKCD, and / or S20 of STK10, and / or S13 of STK10, and / or S144 of PAK1, and / or S141 of PAK2.

[0048] Suitably, the panel of phosphorylation sites may consist of phosphorylation sites on MAPK1 (e.g. at Y187 and / or T185 of MAPK1, and / or at T202 or Y204 of MAPK3, and / or at S21 of GSK3A); Step (a) may include analyzing the data to determine whether a panel of phosphopeptides is phosphorylated in the leukemic cells, and determining an advanced differentiation state if the panel of phosphopeptides is phosphorylated in the cells.

[0049] The data recording the classification of leukemic cells under the French-American-British (FAB) classification system may include any type of data indicative of the FAB classification of leukemic cells. If the leukemic cells are classified as M4, M4eos, or M5 (preferably, if the leukemic cells are classified as M4), they may be determined to be in an advanced differentiation state.

[0050] The methods of the present invention allow for the effective identification of AML patients suitable for kinase inhibitor therapy based on the differentiation status of the patient's leukemic disease. Although various types of kinase inhibitors have already been proposed as candidates for use in AML therapy, there has been no disclosure or suggestion to date that the differentiation state of a patient's leukemia may in any way indicate suitability for kinase inhibitor therapy. Thus, the present invention provides a novel and unobvious grouping of AML patients suitable for kinase inhibitor therapy. As demonstrated by specific examples, this grouping is highly selective with regard to AML patients responding to kinase inhibitor therapy.

[0051] Thus, according to yet another aspect, the present invention provides a kinase pathway inhibitor for use in a method of treating acute myeloid leukemia in a patient, the kinase pathway inhibitor inhibiting a kinase signaling pathway involved in cell proliferation or cell survival, the patient having leukemia cells in an advanced differentiation state. Preferably, the differentiation state of the leukemic cells may be determined according to the methods of the invention described herein.

[0052] Kinase pathway inhibitors are agents such as small molecules or antibodies that block the activity of a kinase pathway. Kinase pathway inhibitors can include inhibitors of enzymes and kinase pathway signaling molecules (eg, kinases, phosphatases, and G proteins). Suitably, the kinase pathway inhibitor may be a kinase inhibitor, which is an agent that blocks the kinase activity of a protein kinase. Such agents are known and widely available in the art. The inhibitory ability of a kinase inhibitor can be evaluated by determining the activity of the kinase before and after incubation with a candidate compound. Kinase profiling methods for identifying kinase inhibitors are also widely available in the art, so that the skilled artisan has at his disposal a wide range of kinase inhibitors for use in the present invention. One assay that can be used to identify agents that can inhibit a particular kinase is the radioactive filter binding assay using 33P ATP, as described in Hastie, et al., 2006. Nat Protoc. 2006;1(2):968-71; Bain, et al., 2007. Biochem J. 2007 Dec 15;408(3):297-315. The method is sensitive, accurate, and provides a direct measurement of activity, so that results can be directly compared between samples.

[0053] Preferably, the kinase pathway inhibitor can inhibit any one or more of the following: FLT3 pathway, PKC pathway, RAS-RAF-MEK-ERK pathway, PI3K-AKT-MTOR-S6K pathway, PAK pathway, JAK-STAT pathway, CAMKK pathway, or any parallel kinase signaling pathway. For example, the kinase pathway inhibitor can be a kinase inhibitor that inhibits one or more of, for example, PKC, PAK, RAF, MEK, ERK, P13K, AKT, MTOR, S6K, STAT5, CAMKK, SYK, LYN, JAK, RTK, ALK, CDK, and BTK.

[0054] In some embodiments, the kinase pathway inhibitor is (a) APS-2-79, AZ 628, AZD8330, BI-847325, binimetinib, BIX 02188, CEP-32496, cobimetinib, dabrafenib, DEL-22379, ERK5-IN-1, FR a MEK inhibitor selected from 180204, GDC-0623, GDC-0994, HA15, honokiol, PD0325901, PD184352, PD318088, PD98059, pimasertib, PLX7904, refametinib, RO5126766, SC1, SCH772984, SCH772984, selumetinib, SGX-523, SL-327, sorafenib, TAK733, trametinib, U0126, U0126, ulixertinib, vandetanib, vemurafenib, VX-11e, and XMD8-92; or (b) AMG 925, Amuvatinib, AZD2932, Cabozantinib, Dovitinib, ENMD-2076, ENMD-2076 L-(+)-Tartaric Acid, G-749, KW-2449, Midostaurin, Pacritinib, Quizartinib, R406, Tandutinib, TCS 359, TG101209, and UNC2025; or an RTK inhibitor selected from imatinib, lenvatinib, lucitanib, sunitinib, osimertinib, erlotinib, gefitinib, dasatinib, nilotinib, lapatinib, pazopanib, ruxolitinib, ponatinib, cabozantinib, regorafenib, bosutinib, axitinib, afatinib, and nintedanib; or (c) a PKC inhibitor selected from enzastaurin, bisindolylmaleimide I, Daphnetin, dequalinium chloride, Go6983, Go6976, LY333531 HCl, Ro 31-8220 mesylate, sotrastaurin, and staurosporine; or (d) a PAK inhibitor selected from FRAX1013, FRAX486, FRAX597, IPA-3, and PF3758309; or (e) 3-Methyladenine, A66, A-674563, Afuresertib, Akti-1 / 2, Alpelisib, AMG319, Apitolisib, AS-252424, AS-604850, AS-605240, AT13148, AT7867, AZD1208 , AZD5363, AZD6482, AZD8055, AZD8186, AZD8835, BGT226, BI-78D3, Buparlisib, CAY10505, CC-223, CCT128930, CH5132799, Copanlisib, CP-466722, CPI-360, CUDC-907, CX-6258 HCl, CZ415, CZC24832, Dactolisib, Duvelisib, ETP-46464, Everolimus, GDC-0084, GDC-0349, Gedatolisib, GNE-317, GSK1059615, GSK2269557, GSK2292767, GSK2636771, GSK690693, HS-173, IC-87114, Idelalisib, INK 128, Ipatasertib, KU-0060648, KU-0063794, KU-55933, LTURM34, LY294002, LY3023414, MHY1485, Miltefosine, Miransertib, MK-2206, NU7026, NU7441, Omipalisib, OSI-027, Palomid 529, Perifosine, PF-0469 1502, PF-4989216, PHT-427, PI-103, PI-3065, Pictilisib, PIK-293, PIK-294, PIK-90, PIK-93, Pilaralisib, PIM447, Piperlongumine, PKI-402, PP121, Rapamycin, Ridaforolimus, SAR405, SC79, SGI-1776, SIS3, SKIa PI3K / AKT / MTOR inhibitor selected from II, SRPIN340, Tacrolimus, Taselisib, Temsirolimus, TG100-115, TG100713, TGR-1202, TIC10, TIC10 analogs, Torin 1, Torin 2, Torkinib, Triciribine, Uprosertib, VE-821, Vistusertib, Voxtalisib, VPS34-IN1, VS-5584, WAY-600, Wortmannin, WYE-125132, WYE-354, WYE-687, XL147 analogs, XL388, Zotarolimus, and ZSTK474; or (f) an ALK inhibitor selected from ceritinib, brigatinib, crizotinib, and alecitinib; or (g) a CDK inhibitor selected from palbociclib and ribociclib; or (h) a JAK inhibitor, such as tofacitinib; or (i) BTK inhibitors such as ibrutinib The kinase inhibitor may be

[0055] Kinase pathway inhibitors include, for example, afatinib, alexitinib, alpelisib, axitinib, bosutinib, brigatinib, buparlisib, cabozantinib, ceritinib, cobimetinib, copanlisib, crizotinib, dabrafenib, dasatinib, dequalinium chloride, duvelisib, erlotinib, everolimus, gefitinib, ibrutinib, idelalisib, imatinib, lapatinib, lenvatinib, and mirabegron. It can be one of lutefosine, nilotinib, nintedanib, osimertinib, pacritinib, palbociclib, pazopanib, ponatinib, quizartinib, radaforolimus, rapamycin, regorafenib, ribociclib, ruxolitinib, selumetinib, sorafenib, sunitinib, temsirolimus, tofacitinib, vandetanib, vemurafenib, and zotarolimus.

[0056] In some preferred embodiments, the kinase pathway inhibitor is MEK inhibitors, for example, APS-2-79, AZ 628, AZD8330, BI-847325, binimetinib, BIX 02188, CEP-32496, cobimetinib, dabrafenib, DEL-22379, ERK5-IN-1, FR 180204, GDC-0623, GDC-0994, HA15, honokiol, PD0325901, PD184352, PD318088, PD98059, pimasertib, PLX7904, refametinib, RO5126766, SC1, SCH772984, SCH772984, selumetinib, SGX-523, SL-327, sorafenib, TAK733, trametinib, U0126, U0126, ulixertinib, vandetanib, vemurafenib, VX-11e, or XMD8-92; or FLT3 / PKC inhibitors, such as midostaurin; or PAK inhibitors, e.g., FRAX1013, FRAX486, FRAX597, IPA-3, and PF3758309; or P13K / AKT / MTOR, e.g., 3-methyladenine, A66, A-674563, afuresertib, Akti-1 / 2, alpelisib, AMG319, apitolisib, AS-252424, AS-604850, AS-605 240, AT13148, AT7867, AZD1208, AZD5363, AZD6482, AZD8055, AZD8186, AZD8835, BGT226, BI-78D3, buparlisib, CAY10505, CC-223, CCT128930, CH5132799, copanlisib, CP-466722, CPI-360, CUDC-907, CX-6258 HCl, CZ415, CZC24832, dactolisib, duvelisib, ETP-46464, everolimus, GDC-0084, GDC-0349, gedatolisib, GNE-317, GSK1059615, GSK2269557, GSK2292767, GSK2636771, GSK690693, HS-173, IC-87114, idelalisib, INK 128, Ipatasertib, KU-0060648, KU-0063794, KU-55933, LTURM34, LY294002, LY3023414, MHY1485, Miltefosine, Miransertib, MK-2206, NU7026, NU7441, Omipalisib, OSI-027, Palomido 529, Perifosine, PF-0469150 2, PF-4989216, PHT-427, PI-103, PI-3065, pictilisib, PIK-293, PIK-294, PIK-90, PIK-93, piralalisib, PIM447, piperlongumine, PKI-402, PP121, rapamycin, ridaforolimus, SAR405, SC79, SGI-1776, SIS3, SKIII, SRPIN340, tacrolimus, taselisib, temsirolimus, TG100-115, TG100713, TGR-1202, TIC10, TIC10 analogues, torin 1, torin 2, torquinib, triciribine, uprosertib, VE-821, bistusertib, voxitalisib, VPS34-IN1, VS-5584, WAY-600, wortmannin, WYE-125132, WYE-354, WYE-687, XL147 analogues, XL388, zotarolimus, or ZSTK474. Suitably, the kinase pathway inhibitor may be trametinib, or midostaurin, or PF 3758309.

[0057] In a particularly preferred embodiment of the invention, the kinase pathway inhibitor is a MEK inhibitor, an FLT3 / PKC inhibitor, or a PAK inhibitor, and step (a) comprises: (i) determining the differentiation state of the patient's leukemia by analyzing data on phosphorylation of one or more phosphorylation sites in MAPK1 and / or MAPK3 in leukemia cells obtained from the patient; and / or (ii) determining the differentiation state of the patient's leukemia by analyzing data on the surface expression on leukemia cells obtained from the patient of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; High levels of phosphorylation at one or more phosphorylation sites and / or the presence of said group of CD markers on said leukemic cells is indicative of an advanced differentiation state.

[0058] The one or more phosphorylation sites in MAPK1 may include Y187 and / or T185. The one or more phosphorylation sites in MAPK3 may include T202 and / or Y204.

[0059] As demonstrated by the experimental data described herein, the inventors have discovered that the present invention provides an accurate test for identifying AML patients who will respond to treatment with an FLT3 / PKC pathway inhibitor, such as midostaurin.

[0060] Thus, in a preferred embodiment, the kinase pathway inhibitor is an FLT3 / PKC pathway inhibitor, such as midostaurin, and step (a) comprises: (i) by analyzing data regarding phosphorylation of phosphorylation sites in one or more of GSK3A, PRKCA, PRKCB, PRKCD, STK10, PAK1, PAK2MAPK1, and / or MAPK3 in leukemic cells obtained from the patient; and / or (ii) by analyzing data on the surface expression on leukemic cells obtained from said patient of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD4PKC5, CD64, CD123, and HLA-DR; determining the differentiation state of the patient's leukemia; Either high levels of phosphorylation in leukemic cells of GSK3A, PRKCA, PRKCB, PRKCD, STK10, PAK1, PAK2MAPK1, and / or MAPK3; or expression by the leukemic cells of said group of CD markers is indicative of an advanced differentiation state.

[0061] In these embodiments, the data regarding phosphorylation of one or more phosphorylation sites in GSK3A, PRKCA, PRKCB, PRKCD, STK10, PAK1, PAK2MAPK1, and / or MAPK3 may include data regarding phosphorylation of GSK3A at pS21, and / or phosphorylation of PRKCD at Y313, pT507, pT295, pT218, and / or pS664, and / or phosphorylation of STK10 at pS20 and / or pS13, and / or phosphorylation of PAK1 at pS144, and / or phosphorylation of PAK2 at pS141, and / or phosphorylation of MAPK1 at Y187 and / or T185, and / or phosphorylation of MAPK3 at T202 and / or Y204.

[0062] The present invention also provides a highly accurate protocol for identifying AML patients who will respond to treatment with a RAS-RAF-MEK-ERK pathway inhibitor, such as trametinib. The present inventors have found that leukemia cells in advanced differentiation states are highly sensitive to treatment with such inhibitors. Such cells often have activating mutations in NRAS, KRAS, HRAS, and / or BRAF. Thus, testing for the presence of such activating mutations complements the present protocol.

[0063] Thus, in some preferred embodiments of the first aspect of the invention, the kinase pathway inhibitor is a RAS-RAF-MEK-ERK inhibitor, such as trametinib; Step (a) further comprises determining the mutation status of NRAS, KRAS, HRAS, or BRAF in the leukemia cells obtained from the patient by analyzing data regarding the genotype of the leukemia cells; Step (b) involves predicting that the patient's acute myeloid leukemia may be effectively treated with the kinase pathway inhibitor if the differentiation state of the leukemia is advanced or if the leukemia cells have an activating mutation in any one of NRAS, KRAS, HRAS, or BRAF.

[0064] In some preferred embodiments of the second aspect of the invention, the kinase pathway inhibitor is a RAS-RAF-MEK-ERK inhibitor, such as trametinib; step (a) further comprises determining the mutational status of NRAS, KRAS, HRAS, or BRAF in leukemic cells obtained from the patient by analyzing data regarding the genotype of the leukemic cells, and step (b) comprises treating the patient with the kinase pathway inhibitor if the differentiation state of the leukemic is progressing or if the leukemic cells have an activating mutation in any one of NRAS, KRAS, HRAS, or BRAF.

[0065] In some preferred embodiments of the third aspect of the invention, the kinase pathway inhibitor is a RAS-RAF-MEK-ERK inhibitor, such as trametinib; Step (a) further comprises determining the mutation status of NRAS, KRAS, HRAS, or BRAF in the leukemia cells obtained from the patient by analyzing data regarding the genotype of the leukemia cells; Step (b) comprises identifying any one or more patients having leukemia in an advanced differentiation state and / or having leukemia with an activating mutation in any one of NRAS, KRAS, HRAS, or BRAF, and determining that said one or more patients may be suitable for effective treatment with the kinase pathway inhibitor.

[0066] In some preferred embodiments of the fourth aspect of the invention, the kinase pathway inhibitor is a RAS-RAF-MEK-ERK inhibitor, such as trametinib; Step (a) further comprises determining the mutation status of NRAS, KRAS, HRAS, or BRAF in the leukemia cells obtained from the patient by analyzing data regarding the genotype of the leukemia cells; Step (b) comprises treating the patient with an inhibitor of the kinase pathway if the differentiation state of the leukemia is progressing or if the leukemia cells have an activating mutation in any one of NRAS, KRAS, HRAS, or BRAF.

[0067] In these preferred embodiments, an activating mutation in NRAS, KRAS, HRAS, or BRAF is a mutation that has the effect of constitutively switching the protein "on." Such mutations may include, for example: NRAS: G12S(c.34G>A) G12C(c.34G>T) G12R(c.34G>C) G12V(c.35G>A) G12A(c.35G>C) G12D(c.35G>A) G13A(c.38G>C) G13V(c.38G>T) G13R(c.37G>C) G13C(c.37G>T) G13S(c.37G>A) G13D(c.38G>A) Q61E(c.181C>G) Q61H(c.183A>T) Q61H(c.183A>C) Q61L(c.182A>T) Q61L(c.182_183AA>TG) Q61K(c.181C>A) Q61P(c.182A>C) Q61R(c.182A>G) Q61R(c.182_183AA>GG) Q61Q(c.183A>G) BRAF: V600E(c.1799T>A) V600K(c.1798_1799GT>AA) V600M(c.1798G>A) V600D(c.1799_1800TG>AT) V600R(c.1798_1799GT>AG) V600G(c.1799T>G) V600E'(c.1799_1800TG>AA)V600A(c.1799_T>C) G469A(c.1406G>A) G469V(c.1406G>T) D594G(c.1781A>G) D594V(c.34G>A) L597(c.1789C) L597(c.1790T) KRAS: G12D(c.34G) G12(c.35G) G13(c.37G) G12(c.38G) Q61(c.181C) Q61(c.182A) Q61(c.183A) A146(c.436G) A146(c.437C) HRAS: G12V(c.34G) G12(c.35G) G13(c.37G) G13(c.38G) Q61(c.181C) Q61(c.182A) Q61(c.183G)

[0068] In these embodiments, the data regarding the genotype of the leukemic cells may include any information that would allow one of skill in the art to infer the presence or absence of activating mutations in NRAS, KRAS, HRAS, and / or BRAF. This data may include, but is not limited to: The sequences of the NRAS, KRAS, HRAS, and / or BRAF genes in the leukemic cells, the encoded proteins or the sequences of each encoded protein expressed by the leukemic cells, or data recording the presence or absence of activating mutations in NRAS, KRAS, HRAS, and / or BRAF in the leukemic cells. In some embodiments, the data has already been collected and recorded, and step (a) comprises obtaining the data for analysis. In other embodiments, step (a) further comprises collecting and recording the data for analysis. The data may be readily collected and recorded according to techniques and protocols known in the art and as exemplified herein.

[0069] In these embodiments, step (a) comprises: (i) determining the differentiation state of the patient's leukemia by analyzing data on phosphorylation of one or more phosphorylation sites in MAPK1 or MAPK3 in leukemia cells obtained from the patient; and / or (ii) determining the differentiation state of the patient's leukemia by analyzing data on the surface expression on leukemia cells obtained from the patient of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; It may include, High levels of phosphorylation at one or more phosphorylation sites and / or the presence of said group of CD markers on said leukemic cells is indicative of an advanced differentiation state.

[0070] The present inventors have also discovered that leukemia cells that have activating mutations in FLT3, or that exhibit activation of the FLT3-driven pro-survival kinase signalling pathway that acts in parallel with the RAS-RAF-MEK-ERK pathway, or that have high levels of phosphorylation on certain phosphomarkers, as identified below, may be resistant to treatment with MEK pathway inhibitors. It is believed that mutations in FLT3 and / or activation of the parallel FLT3-driven pro-survival signalling pathway may provide cells with an alternative survival mechanism despite inhibition of the RAS-RAF-MEK-ERK pathway.

[0071] In some preferred embodiments of the invention, the kinase pathway inhibitor is a RAS-RAF-MEK-ERK pathway inhibitor, such as a MEK inhibitor (e.g., trametinib), and step (a) comprises: (i) determining the FLT3 mutation status in leukemic cells obtained from a patient by analyzing data relating to the genotype of said leukemic cells; and / or (ii) determining activation in said leukemia cells of a FLT3-driven kinase signaling pathway involved in cell proliferation or cell survival other than the RAS-RAF-MEK-ERK pathway, such as the PKC pathway, the PI3K-AKT-MTOR-S6K pathway, the PAK pathway, the JAK-STAT pathway, or the CAMKK pathway, by analyzing data on kinase pathway activity markers in said leukemia cells; and / or (iii) determining the level of phosphorylation of one or both of TOP2A and / or KDM5C in the leukemia cells by analyzing the data regarding the phosphorylation of TOP2A and / or KDM5C in the leukemia cells; Further includes:

[0072] In such an embodiment according to the first aspect of the invention, step (b) comprises the steps of: (i) the differentiation state of the leukemia is advanced; and (ii) predicting that the patient's acute myeloid leukemia can be effectively treated with a RAS-RAF-MEK-ERK pathway inhibitor if FLT3 in the leukemia cells does not have an activating mutation, or if the FLT3-driven kinase signaling pathway is not activated in the leukemia cells, or if TOP2A and / or KDM5C are phosphorylated at low levels in the leukemia cells. In such an embodiment according to the second aspect of the invention, step (b) comprises: (i) the differentiation state of the leukemia is advanced; and (ii) if FLT3 in the leukemia cells does not have an activating mutation, or the FLT3-driven kinase signaling pathway is not activated in the leukemia cells, or TOP2A and / or KDM5C are phosphorylated at low levels in the leukemia cells, this may include treating the patient with a RAS-RAF-MEK-ERK pathway inhibitor. In such an embodiment of the third aspect of the invention, step (b) comprises: (i) the leukemia cells have an advanced differentiation state; and (ii) identifying one or more patients whose leukemia cells do not have an activating mutation in FLT3, or whose FLT3-driven kinase signaling pathway is not activated in the leukemia cells, or whose TOP2A and / or KDM5C are phosphorylated at low levels in the leukemia cells, as suitable for effective treatment with a RAS-RAF-MEK-ERK inhibitor. In such an embodiment of the fourth aspect of the invention, step (b) comprises: (i) the differentiation state of the leukemia is advanced; and (ii) If FLT3 in the leukemia cells does not have an activating mutation, or the FLT3-driven kinase signaling pathway is not activated in the leukemia cells, or TOP2A and / or KDM5C are phosphorylated at low levels in the leukemia cells, this may include treating the patient with a RAS-RAF-MEK-ERK inhibitor.

[0073] Accordingly, some preferred related embodiments of the first aspect of the invention include (a)(i) determining the leukemic differentiation status of the patient by analyzing data regarding the genotype of the leukemic cells and / or determining the NRAS, KRAS, HRAS, or BRAF mutation status in the leukemic cells obtained from the patient; and (ii) determining the mutation status of FLT3 in leukemic cells obtained from a patient by analyzing data on the genotype of said leukemic cells, and / or determining activation in said leukemic cells of FLT-3-driven kinase signaling pathways involved in cell proliferation or cell survival other than the RAS-RAF-MEK-ERK pathway by analyzing data on activation markers of FLT3-driven kinase signaling pathways in said leukemic cells; and / or determining the level of phosphorylation of one or both of TOP2A and / or KDM5C in said leukemic cells by analyzing data on phosphorylation of TOP2A and / or KDM5C in said leukemic cells; (b)(i) the differentiation state of the leukemia is advanced or the leukemia cells have an activating mutation in any one of NRAS, KRAS, HRAS, or BRAF; and (ii) if FLT3 in the leukemia cells does not have an activating mutation, or the FLT3-driven kinase signaling pathway is not activated, or TOP2A and / or KDM5C are phosphorylated at low levels in the leukemia cells, predicting that this patient's acute myeloid leukemia can be effectively treated with said RAS-RAF-MEK-ERK pathway inhibitor may include.

[0074] Accordingly, some preferred embodiments of the second aspect of the invention include (a)(i) determining the leukemic differentiation status of the patient by analyzing data regarding the genotype of the leukemic cells and / or determining the NRAS, KRAS, HRAS, or BRAF mutation status in the leukemic cells obtained from the patient; and (ii) determining the mutation status of FLT3 in leukemic cells obtained from a patient by analyzing data on the genotype of said leukemic cells, and / or determining activation in said leukemic cells of FLT-3-driven kinase signaling pathways involved in cell proliferation or cell survival other than the RAS-RAF-MEK-ERK pathway by analyzing data on activation markers of FLT3-driven kinase signaling pathways in said leukemic cells; and / or determining the level of phosphorylation of one or both of TOP2A and / or KDM5C in said leukemic cells by analyzing data on phosphorylation of TOP2A and / or KDM5C in said leukemic cells; (b)(i) the differentiation state of the leukemia is advanced or the leukemia cells have an activating mutation in any one of NRAS, KRAS, HRAS, or BRAF; and (ii) if FLT3 in the leukemia cells does not have an activating mutation, or the FLT3-driven kinase signaling pathway is not activated, or TOP2A and / or KDM5C are phosphorylated at low levels in the leukemia cells, Treating the patient with the RAS-RAF-MEK-ERK pathway inhibitor. may include.

[0075] Accordingly, some preferred embodiments of the third aspect of the invention include (a) with respect to each patient: (i) determining the leukemic differentiation status of the patient by analyzing data regarding the genotype of the leukemic cells and / or determining the NRAS, KRAS, HRAS, or BRAF mutation status in the leukemic cells obtained from the patient; and (ii) determining the mutation status of FLT3 in leukemic cells obtained from a patient by analyzing data on the genotype of said leukemic cells, and / or determining activation in said leukemic cells of FLT-3-driven kinase signaling pathways involved in cell proliferation or cell survival other than the RAS-RAF-MEK-ERK pathway by analyzing data on activation markers of FLT3-driven kinase signaling pathways in said leukemic cells; and / or determining the level of phosphorylation of one or both of TOP2A and / or KDM5C in said leukemic cells by analyzing data on phosphorylation of TOP2A and / or KDM5C in said leukemic cells; and (b) With respect to any one or more patients: (i) the differentiation state of the leukemia is advanced or the leukemia cells have an activating mutation in any one of NRAS, KRAS, HRAS, or BRAF; and (ii) if FLT3 in the leukemia cells does not have an activating mutation, or the FLT3-driven kinase signaling pathway is not activated, or TOP2A and / or KDM5C are phosphorylated at low levels in the leukemia cells, Identifying one or more patients as having leukemia amenable to effective treatment with said RAS-RAF-MEK-ERK pathway inhibitor. may include.

[0076] Accordingly, some preferred embodiments of the fourth aspect of the invention include (a)(i) determining the leukemic differentiation status of the patient by analyzing data regarding the genotype of the leukemic cells and / or determining the NRAS, KRAS, HRAS, or BRAF mutation status in the leukemic cells obtained from the patient; and (ii) determining the mutation status of FLT3 in leukemic cells obtained from the patient by analyzing data on the genotype of the leukemic cells, and / or determining activation in the leukemic cells of an FLT-3-driven kinase signaling pathway involved in cell proliferation or cell survival other than the RAS-RAF-MEK-ERK pathway by analyzing data on activation markers of the FLT3-driven kinase signaling pathway in the leukemic cells; and / or determining phosphorylation at one or more phosphorylation sites in one or both of TOP2A and / or KDM5C in the leukemic cells by analyzing data on phosphorylation of TOP2A and / or KDM5C in the leukemic cells; and (b)(i) the differentiation state of the leukemia is advanced or the leukemia cells have an activating mutation in any one of NRAS, KRAS, HRAS, or BRAF; and (ii) FLT3 in the leukemia cells does not have an activating mutation, or the FLT3-driven kinase signaling pathway is not activated, or TOP2A and / or KDM5C are phosphorylated at low levels in the leukemia cells; Treating the patient with the RAS-RAF-MEK-ERK pathway inhibitor. may include.

[0077] In these preferred embodiments, activating mutations in FLT3 are those that have the effect of constitutively switching the FLT3 protein "on". For example, such mutations may include internal tandem duplications (ITDs) of the juxtamembrane domain, or point mutations that usually involve the tyrosine kinase domain (e.g., at D835). Said data on the genotype of leukemia cells can include any information that allows a person skilled in the art to infer the presence or absence of activating mutations in FLT3. This data can include, but is not limited to: the sequence of the FLT3 gene in leukemia cells, the sequence of the FLT3 protein expressed by leukemia cells, or data recording the presence or absence of activating mutations in FLT3 in leukemia cells. A person skilled in the art can easily collect and interpret said data according to techniques and protocols well known in the art.

[0078] In these preferred embodiments, the step of determining activation in leukemia cells of a FLT3-driven kinase signaling pathway involved in cell proliferation or cell survival other than the RAS-RAF-MEK-ERK pathway may include determining activation of multiple types of FLT3-driven kinase signaling pathways. Preferably, the or each FLT3-driven kinase signaling pathway may be selected from the PKC pathway, the PI3K-AKT-MTOR-S6K pathway, the PAK pathway, the JAK-STAT pathway, or the CAMKK pathway. Preferably, the FLT3-driven kinase signaling pathway can be the JAK-STAT pathway, the PI3K-AKT-MTOR-S6K pathway, or the CAMKK pathway.Suitably, the FLT3-driven kinase signaling pathway can be the JAK-STAT (STAT5) pathway.

[0079] Said activity marker of the FLT3-driven kinase signaling pathway may include any marker that can be used to identify activation of the FLT3-driven kinase signaling pathway. These may include any kinase, phosphatase, phospholipase, integrin, signal transduction regulator, G protein, transmembrane receptor, phosphopeptide, and / or other kinase signaling molecule that is selectively activated in the FLT3-driven kinase signaling pathway, or any molecule that is selectively phosphorylated in the FLT3-driven kinase signaling pathway (phosphomarker). Advantageously, the activity marker of the FLT3-driven kinase signaling pathway may comprise one or more phosphomarkers, and the data on the activity marker may comprise data on the phosphorylation of the one or more phosphomarkers, with a high level of phosphorylation of the one or more phosphomarkers indicating that the FLT3-driven kinase signaling pathway is activated.

[0080] The one or more phosphomarkers may include, for example, one or more phosphorylation sites of the proteins STAT5A and / or CAMKK1, for example, phosphorylation sites at S780 and / or S128 of STAT5A and phosphorylation sites at S548 of CAMKK1. These phosphorylation sites are selectively phosphorylated in the FLT3-driven kinase signaling pathway. Thus, data regarding activity markers may include data regarding the level of phosphorylation of either or both of STAT5A and CAMKK1 (e.g., the level of phosphorylation of STAT5A at S780 and / or S128 and / or the level of phosphorylation of CAMKK1 at S548), with high levels of phosphorylation indicating activation of the FLT3-driven kinase signaling pathway.

[0081] In these preferred embodiments, the data regarding phosphorylation of TOP2A and / or KDM5C in leukemic cells may include data regarding phosphorylation of TOP2A and / or KDM5C (e.g., data regarding phosphorylation of TOP2A at S1213 and / or phosphorylation of KDM5C at S317).

[0082] The present invention also contemplates the use of surrogate phosphomarkers of the FLT3-driven kinase signaling pathway (e.g., the STAT5 pathway and / or the CAMKK pathway), which can similarly be used to determine activation of the FLT3-driven kinase signaling pathway.

[0083] Data regarding an activity marker may include any information that allows a person skilled in the art to infer the activation of the activity marker (e.g., the expression or activation of the activity marker, e.g., the level of phosphorylation of the activity marker). Such data may include, for example, LC-MS / MS data. In some embodiments, the data has already been collected and recorded, and step (a)(ii) comprises obtaining the data for analysis. In other embodiments, step (a)(ii) further comprises collecting and recording the data for analysis. The data may be collected and recorded without difficulty according to techniques and protocols known in the art and as exemplified herein (e.g., by LC-MS / MS or by immunochemical techniques).

[0084] In these embodiments, the differentiation state of the leukemic cells may be determined according to any of the method steps described herein. However, preferably, the step of determining the differentiation state of the leukemic cells comprises: (i) analyzing data regarding phosphorylation of one or more phosphorylation sites in MAPK1 or MAPK3 in leukemia cells obtained from the patient; and / or (ii) determining the differentiation state of the patient's leukemia by analyzing data on the surface expression on leukemia cells obtained from the patient of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; It may include, High levels of phosphorylation at one or more phosphorylation sites and / or the presence of said group of CD markers on said leukemic cells is indicative of an advanced differentiation state.

[0085] The present invention is of particular interest with respect to kinase pathway inhibitors that are approved for the treatment of AML or may soon be approved for the treatment of AML. These include the FLT3 / PKC pathway inhibitor midostaurin, and the RAS-RAF-MEK-ERK pathway inhibitor trametinib. As demonstrated herein, the present invention provides a significantly improved protocol for identifying patients who will respond to treatment with these kinase pathway inhibitors. The availability of an accurate companion diagnostic test to identify potentially responsive patients would be of significant therapeutic and clinical benefit, as it would help ensure that patients who respond to treatment are identified and can benefit from this treatment, and that non-responsive patients do not receive treatment unnecessarily.

[0086] Thus, the present invention provides midostaurin for use in a method of treating acute myeloid leukemia in a patient, the patient comprising: (i) surface expression on the patient's leukemic cells of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; and / or (ii) high levels of phosphorylation of any one or more of the phosphorylation sites on GSK3A, PRKCA, PRKCB, PRKCD, STK10, PAK1, PAK2, MAPK1, and / or MAPK3 in the patient's leukemic cells; The patients have leukemia with an advanced differentiation state as defined by:

[0087] Specifically, the present invention provides midostaurin for use in a method of treating acute myeloid leukemia in a patient, the patient comprising: (i) surface expression on the patient's leukemic cells of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; and / or (ii) The following: pS21 of GSK3A; PRKCD pY313, pT507, pT295, pT218, and / or pS664; pS20 and / or pS13 of STK10; PAK1 pS144; PAK2 pS141; Y187 and / or T185 of MAPK1; and T202 and / or Y204 in MAPK3 High levels of phosphorylation in the patient's leukemia cells The patients have leukemia with an advanced differentiation state as defined by:

[0088] The present invention further provides trametinib for use in a method of treating acute myeloid leukemia in a patient, the patient comprising: (a)(i) high levels of phosphorylation of one or more phosphorylation sites in MAPK1 and / or MAPK3 in the patient's leukemic cells; and / or (ii) surface expression on the patient's leukemic cells of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; and / or (iii) an activating mutation in any one of NRAS, KRAS, HRAS, or BRAF in the patient's leukemia cells; and (b)(i) high levels of phosphorylation of one or more of STAT5A, TOP2A, KDM5C, and CAMKK1 in the leukemia cells; and / or (ii) the absence of any activating mutations in FLT3 in this patient's leukemic cells; These patients have leukemias with an advanced differentiation state defined by and hypoactivation of FLT3-driven survival pathways.

[0089] In particular, the present invention provides trametinib for use in a method of treating acute myeloid leukemia in a patient, the patient comprising: (a)(i) surface expression on the patient's leukemic cells of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; and / or (ii) high levels of phosphorylation in the patient's leukemic cells of any one or more of Y187 and / or T185 of MAPK1; and T202 and / or Y204 of MAPK3; (iii) activating mutations in one or more of NRAS, KRAS, HRAS, or BRAF in the patient's leukemia cells; and (b)(i) high levels of phosphorylation at one or more of pS780 and pS128 of STAT5A, pS548 of CAMKK1, pS1213 of TOP2A, and pS317 of KDM5C; and / or (ii) the absence of any activating mutations in FLT3 in this patient's leukemic cells; The patients have leukemia with an advanced differentiation state as defined by:

[0090] The present invention provides a method of treating acute myeloid leukemia in an individual patient suffering from acute myeloid leukemia, comprising: (a) obtaining a biological sample from the patient, the biological sample comprising leukemia cells; (b) determining by in vitro clinical testing that the differentiation state of the leukemia cells in the sample obtained in (a) has progressed; and (c) if the differentiation state of the leukemic cells is progressing, administering to the patient a kinase pathway inhibitor, which inhibits a signal transduction pathway involved in cell proliferation or cell survival. The present invention further provides a method comprising:

[0091] The biological sample can be a peripheral blood sample or a bone marrow sample.The kinase pathway inhibitor can be selected from a MEK pathway inhibitor, a FLT3 / PKC pathway inhibitor, and a PAK pathway inhibitor.

[0092] In this aspect of the invention, step (b) may comprise detecting morphological and / or cytochemical characteristics of leukemic cells in a sample obtained from the patient, where M4 classification under the French-American-British (FAB) classification system indicates an advanced differentiation state.

[0093] The step of detecting morphological and / or cytochemical characteristics of leukemic cells may include preparing the cells for microscopic analysis and visually observing the cells under a light microscope to detect morphological signs of differentiation; and / or assaying the behavior, activity, or response of the cells to specific conditions or test reagents (e.g., Sudan Black B, and / or peroxidase, and / or specific or non-specific esterases).

[0094] Optionally, step (b) may comprise performing an in vitro assay to detect the expression level of one or more cell surface differentiation markers on the surface of leukemic cells in a sample obtained from the patient, the cell surface differentiation markers being generally expressed or overexpressed on healthy myelomonocytic cells and the cell surface differentiation markers being generally not expressed or overexpressed on undifferentiated myeloblasts, and expression of said one or more cell surface differentiation markers at high levels on the surface of leukemic cells indicating an advanced differentiation state. The assay may be an LC-MS / MS assay or an immunochemical assay, for example a Western blot assay, an ELISA assay, or a reverse phase protein assay.

[0095] The cell surface differentiation markers may include a panel of cell surface marker proteins including one or more of CD3, CD7, CD11b, CD11c (integrin alpha-X, ITAX), CD14, CD15, CD16, CD18 (integrin beta, ITB2), CD19, CD33, CD34, CD35 (CR1), CD38, CD44, CD45, CD64, CD97, CD117, CD123, CD180, CD184, HLA-C (1C02), APOBR, platelet membrane receptor Gi24 (VSIR), and HLA-DR; and / or any cell surface protein expressed in conjunction with said one or more cell surface marker proteins.

[0096] Suitably, the panel of cell surface marker proteins comprises: (i) any one, two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen, or fifteen, or sixteen, or seventeen, or eighteen, or all, of CD11b, CD11c (integrin α-X, ITAX), CD14, CD15, CD16, CD18 (integrin β, ITB2), CD33, CD35 (CR1), CD38, CD44, CD45, CD64, CD97, CD123, CD180, HLA-C (1C02), APOBR, platelet membrane receptor Gi24 (VSIR), and HLA-DR; or (ii) any one, two, three, four, five, six, seven, eight, nine, ten, or all of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; or (iii) any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all of CD3, CD7, CD11b, CD14, CD15, CD16, CD19, CD33, CD34, CD38, CD44, CD45, CD64, CD117, CD123, CD184, and HLA-DR; may include.

[0097] In the method of treatment according to this aspect of the invention, step (b) comprises carrying out an in vitro assay to detect expression, and / or activation, and / or phosphorylation of one or more functional differentiation markers in leukemic cells in a sample obtained from the patient, which functional differentiation markers are generally expressed, overexpressed, activated, and / or phosphorylated in healthy myelomonocytic cells and which functional differentiation markers are generally not expressed, overexpressed, activated, and / or phosphorylated in undifferentiated myeloblasts, and wherein expression, activation, and / or phosphorylation of said one or more functional differentiation markers in leukemic cells indicates an advanced differentiation state. The assay may be an LC-MS / MS assay or an immunochemical assay, for example a Western blot assay, an ELISA assay, or a reverse phase protein assay. The one or more functional differentiation markers may comprise a panel of protein markers including one or more enzymes, integrins, kinases, phosphatases, signal transduction regulators, cytoplasmic proteins and phosphoproteins, membrane proteins and phosphoproteins, e.g., GTPases or cytoplasmic phosphoproteins involved in other forms of cell signaling, which protein markers are generally expressed, overexpressed, and / or activated in healthy myelomonocytic cells and generally not expressed, overexpressed, and / or activated in undifferentiated myeloblasts, and expression and / or activation of the panel of protein markers in leukemic cells indicates an advanced differentiation state.

[0098] Optionally, the panel of protein markers may include any one, two, three, four, or five of lysozyme C (LYZ), neutrophil cytosolic factor 2 (NCF2), myeloid nuclear differentiation antigen (MNDA), AK1C4, and ERG.

[0099] Suitably, the one or more functional differentiation markers may comprise a panel of kinase pathway activity markers including one or more kinases, phosphatases, phospholipases, integrins, signal transduction regulators, G proteins, transmembrane receptors, phosphopeptides and / or other kinase signaling molecules that are generally expressed and / or activated and / or phosphorylated in kinase signaling pathways in healthy myelomonocytic cells but not generally expressed and / or activated and / or phosphorylated in undifferentiated myeloblasts, and expression and / or activation and / or phosphorylation of this panel of kinase pathway activity markers in leukemic cells is indicative of an advanced differentiation state. Specifically, the panel of kinase pathway activity markers may include markers of kinase signaling pathways that are inhibited by kinase pathway inhibitors.

[0100] Panels of kinase pathway activity markers include, for example, FES, PKC and protein kinase C isoforms (e.g., PKC delta (KPCD), PRKCA, PRKCB, and PRKCD), PKA, PAK (e.g., PAK1 and PAK2), STK10, GSK3A, RSK2, RAS, RAF, MEK (e.g., MEK1 (MAP2K1)), ERK (e.g., MAPK3 (ERK1) and MAPK1 (ERK2)), PI3K, AKT (e.g., AKT1), MTOR, S6 kinase, STAT5, CAMKK, SYK (KSYK), LYN, P38A, CDK1, CK2A1, PKACA, IRAK4, PKCB The kinases may include any one, two, three, four, five, six, seven, eight, nine, ten, or more of iso2, Cot, PKCD, PKCA, PKCB, PKCG, PKCH, BRAF, MEK2, PDK1, CDK2, PTN6, D3(PLD3), IQGAP1, GRB2, RHOA, RHOG, and S10AB, as well as any kinases, phosphatases, phospholipases, integrins, signal transduction regulators, G proteins, transmembrane receptors, and / or other kinase signaling molecules that are selectively expressed or activated therewith.

[0101] In some embodiments, the panel of kinase pathway activity markers comprises: (i) any one, two, three, or four of PKC, ERK, PAK1, and p38α; (ii) any one, two, three, four, or five of PKCD, PKCA, PKACA, IRAK4, and CK2A1; or (iii) any one, two, three, four, five, or six of MAPK1, MAPK2, AKT, AKT1S1, MAP2K1, and MAP2K2; may include.

[0102] In some embodiments, the panel of kinase pathway activity markers may include a panel of one or more phosphorylation sites that are generally phosphorylated or generally highly phosphorylated in kinase signaling pathways in healthy myelomonocytic cells, but are generally not phosphorylated or generally not highly phosphorylated in undifferentiated myeloblasts, where phosphorylation or high levels of phosphorylation at this panel of phosphorylation sites in leukemic cells indicates an advanced differentiation state.

[0103] In such an embodiment, the panel of phosphorylation sites comprises: (i) any one, two, three, four, five, six, seven, eight, nine, ten, or more than ten phosphorylation sites listed in Table 1; or (ii) any one, two, three, four, five, six, seven, eight, nine, ten, or more than ten phosphorylation sites listed in Table 2; or (iii) PAK1 at S144, PAK2 at S141, MAPK1 at Y187, MAPK1 at T185, RPS6KA1 at S380, MAPK3 at T202, MAPK3 at Y204, MAP3K3 at S166, SYK at S295 and S297, IRAK3 at S110, PKN1 (379–396+phosphoST), STK10 (447–464+phosphoST), and S410 RIPK3 at T218, PRKCD at T295, PRKCD at Y313, PRKCD at T507, PRKCD at T645, PRKCD at S664, PRKCD at T2638, MARK2 at S535, MAP3K2 at S535, PRKD2 (710-730+phosphoY), NRK at s805, PRKAR2A at S58, ZAK (591-616+phosphoY) ST), MAP4K4 at S900, CDK9 at S347, RPS6KA4 (681-699+2 phospho-ST), MAST3 (1254-1274+ phospho-ST), NEK9 (10-39+ phospho-ST), GSK3A (19-50+ phospho-ST), RPS6KA3 at S369, RIPK2 at S531, AAK1 at T606, TYK2 at Y292, PDPK2 at S214, PRKA or any one, two, three, four, five, six, seven, eight, nine, ten, or more than ten of: A1 (3-8+phosphoST), STK11P at S772, BAZ1B at S1468, CLK1 at S140, MAP4K2 at S328, WNK1 (1996-2021+phosphoST), CDK11A at S271, FES at Y713, and / or TNIK at S769; or (iv) PAK1 at S144, PAK2 at S141, MAPK1 at Y187 and / or T185, and RPS6KA1 at S380; or (v) MAPK1 at Y187, PAK2 at S141, and PRKCD at Y313; or (vi) FES at Y713, MAPK3 at T202 / Y204, MAPK1 at T185 / Y187, PAK1 at S144, MEK1 at S222, PAK2 at S141, and PRKCD at S645; or (vii) GSK3A at S21, and / or PRKCD at T507, T295, T218, Y313, T507 and / or S664, and / or STK10 at S20 and / or S13, and / or PAK1 at S144, and / or PAK2 at S141. (viii) one or more phosphorylation sites in MAPK1 (e.g., MAPK1 at Y187 and / or T185), and MAPK3 at T202 and / or Y204, and GSK3A at S21 may include.

[0104] In the methods of treating acute myeloid leukemia according to the present invention, the kinase pathway inhibitor may inhibit any one or more of the FLT3 pathway, the PKC pathway, the RAS-RAF-MEK-ERK pathway, the PI3K-AKT-MTOR-S6K pathway, the PAK pathway, the JAK-STAT pathway, the CAMKK pathway, or any kinase signaling pathways parallel thereto. Suitably, the kinase pathway inhibitor may be a MEK inhibitor, or a FLT3 / PKC inhibitor, or a PAK inhibitor.

[0105] In such an embodiment, step (b) comprises: (i) performing an in vitro assay to detect and / or quantify phosphorylation of one or more phosphorylation sites in MAPK1 and / or MAPK3 in leukemic cells in a sample obtained from a patient; and / or (ii) performing an in vitro assay to detect and / or quantify surface expression on leukemic cells in a sample obtained from said patient of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; It may include, Phosphorylation or high levels of phosphorylation at the phosphorylation site(s) and / or high levels of expression of said group of CD markers on said leukemic cells is indicative of an advanced differentiation state.

[0106] In the method of treating acute myeloid leukemia according to the present invention, the kinase pathway inhibitor is an FLT3 / PKC pathway inhibitor such as midostaurin, and step (b) comprises: (i) performing an in vitro assay to detect and / or quantify phosphorylation of phosphorylation sites in one or more of GSK3A, PRKCA, PRKCB, PRKCD, STK10, PAK1, PAK2MAPK1, and / or MAPK3 in leukemic cells in a sample obtained from a patient; and / or (ii) performing an in vitro assay to detect and / or quantify surface expression of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD4PKC5, CD64, CD123, and HLA-DR on the surface of leukemic cells in a sample obtained from the patient; It may include, Either phosphorylation or high levels of phosphorylation in the leukemic cells of any one or more of GSK3A, PRKCA, PRKCB, PRKCD, STK10, PAK1, PAK2, MAPK1, and / or MAPK3; or high levels of expression by the leukemic cells of the above groups of CD markers indicates an advanced differentiation state.

[0107] In the method of treating acute myeloid leukemia according to the present invention, the kinase pathway inhibitor is a MEK inhibitor, such as trametinib, and step (b) may further comprise carrying out an in vitro assay to detect the genotype of leukemic cells obtained from the patient, and determining that any one of NRAS, KRAS, HRAS, or BRAF in the leukemic cells in the sample obtained from the patient has an activating mutation.Suitably, the assay may comprise sequencing NRAS, KRAS, HRAS, or BRAF in the leukemic cells in the sample obtained from the patient, thereby identifying the activating mutation in the sequence data obtained.

[0108] In these embodiments, step (b) comprises: (i) performing an in vitro assay to detect phosphorylation of one or more phosphorylation sites in MAPK1 or MAPK3 in leukemic cells in a sample obtained from a patient; and / or (ii) performing an in vitro assay to detect surface expression of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR on leukemic cells in a sample obtained from the patient; It may include, Phosphorylation or high levels of phosphorylation at the phosphorylation site(s) and / or high levels of expression of said group of CD markers on said leukemic cells is indicative of an advanced differentiation state.

[0109] In the method of treating acute myeloid leukemia according to the present invention, the kinase pathway inhibitor is a MEK inhibitor such as trametinib, and step (b) comprises: (i) performing an in vitro assay to detect the genotype of leukemia cells in a sample obtained from a patient and to determine that FLT3 in the leukemia cells does not have an activating mutation; and / or (ii) performing an in vitro assay to detect expression or activation in leukemia cells in a sample obtained from a patient of one or more activation markers of an FLT-3-driven kinase signaling pathway other than the RAS-RAF-MEK-ERK pathway that is involved in cell proliferation or cell survival (e.g., the PKC pathway, the PI3K-AKT-MTOR-S6K pathway, the PAK pathway, the JAK-STAT pathway, or the CAMKK pathway), and determining that the FLT3-driven kinase signaling pathway is not activated in the leukemia cells; and / or (iii) performing an in vitro assay to detect the level of phosphorylation of one or both of TOP2A and / or KDM5C in leukemia cells in a sample obtained from a patient, and determining that TOP2A and / or KDM5C are not phosphorylated or are phosphorylated at a low level in the leukemia cells; It may further include.

[0110] In the method of treating acute myeloid leukemia according to the present invention, wherein the kinase pathway inhibitor is a MEK inhibitor such as trametinib, step (b) comprises: (i) performing an in vitro assay to detect the genotype of leukemic cells in a sample obtained from a patient and determine that the leukemic cells have an activating mutation in any one of NRAS, KRAS, HRAS, or BRAF; and / or determine that the differentiation state of the leukemic cells in the sample obtained from the patient is progressing; and (ii) performing an in vitro assay to detect the genotype of leukemia cells in a sample obtained from the patient and determining that FLT3 in the leukemia cells does not have an activating mutation; and / or performing an assay to detect expression or activity in leukemic cells in a sample obtained from a patient of one or more activation markers of an FLT-3-driven signaling pathway other than the RAS-RAF-MEK-ERK pathway that is involved in cell proliferation or cell survival (e.g., the PKC pathway, the PI3K-AKT-MTOR-S6K pathway, the PAK pathway, the JAK-STAT pathway, or the CAMKK pathway), and determine that the FLT3-driven kinase signaling pathway is not activated in the leukemic cells; and / or performing an assay to detect the level of phosphorylation of one or both of TOP2A and / or KDM5C in leukemic cells in a sample obtained from a patient, and determine that TOP2A and / or KDM5C are not phosphorylated or are phosphorylated at a low level in the leukemic cells. may include.

[0111] Suitably, said activity marker of a FLT3-driven signalling pathway may comprise one or more phosphorylation sites which are selectively phosphorylated by the FLT3-driven kinase signalling pathway. In these embodiments, phosphorylation or high levels of phosphorylation at the phosphorylation site or sites indicates that a FLT3-driven kinase signaling pathway is activated. The one or more phosphorylation sites may include phosphorylation sites in one or both of STAT5A and CAMKK1, for example STAT5A at S780 and / or S128, and / or CAMKK1 at S548.

[0112] Optionally, the step of detecting the level of phosphorylation of one or both of TOP2A and / or KDM5C in leukemia cells may include detecting phosphorylation of TOP2A at S1213 and / or phosphorylation of KDM5C at S317.

[0113] In certain aspects, the present invention provides methods of treating acute myeloid leukemia in an individual patient suffering from acute myeloid leukemia. (a) obtaining a biological sample from the patient, the biological sample comprising leukemia cells; (b)(i) performing an in vitro assay to detect expression on the surface of leukemic cells in a sample obtained from said patient of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; and / or (ii) performing an in vitro assay to detect phosphorylation in the leukemic cells of one or more phosphorylation sites selected from GSK3A at pS21; PRKCD at Y313, pT507, pT295, pT218, and / or pS664 of PRKCD; STK10 at pS20 and / or pS13 of STK10; PAK1 at pS144 of PAK1; PAK2 at pS141 of PAK2; MAPK1 at Y187 and / or T185; and MAPK3 at T202 and / or Y204; and (c) administering midostaurin to the patient for the treatment of acute myeloid leukemia if the group of CD markers is expressed at a high level on the surface of the patient's leukemia cells and / or one or more of the phosphorylation sites in the leukemia cells is phosphorylated or highly phosphorylated. The present invention provides a method comprising:

[0114] In another particular aspect, the present invention provides a method of treating acute myeloid leukemia in an individual patient suffering from acute myeloid leukemia, comprising: (a) obtaining a biological sample from the patient, the biological sample comprising leukemia cells; (b)(i) performing an in vitro assay to detect expression of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR on the surface of leukemic cells in a sample obtained from the patient; and / or (ii) performing an in vitro assay to detect phosphorylation in leukemic cells of one or more MEK-associated phosphorylation sites selected from MAPK1 at Y187 and / or T185, and MAPK3 at T202 and / or Y204; and / or (iii) performing an in vitro assay to genotype the patient's leukemic cells to determine whether an activating mutation in any one or more of NRAS, KRAS, HRAS, or BRAF is present in the leukemic cells; (c)(i) performing an in vitro assay to detect phosphorylation in leukemic cells at one or more marker sites selected from STAT5A at pS780 and / or pS128, CAMKK1 at pS548, TOP2A at pS1213, and KDM5C at pS317 of KDM5C; and / or (ii) performing an in vitro assay to detect the genotype of the patient's leukemia cells to determine whether an activating mutation in FLT3 is present in the leukemia cells; and (d)(A) the group of CD markers are expressed at high levels on the surface of the patient's leukemia cells, and / or one or more of the MEK-associated phosphorylation sites in the leukemia cells are phosphorylated or highly phosphorylated, and / or there is an activating mutation in any one or more of NRAS, KRAS, HRAS, or BRAF in the patient's leukemia cells; and (B) if the marker site is not phosphorylated or is not highly phosphorylated in the patient's leukemia cells, and / or if there is no activating mutation in FLT3 in the patient's leukemia cells, administering trametinib to the patient for the treatment of acute myeloid leukemia. The present invention provides a method comprising:

[0115] The invention will now be described with reference to the embodiments and drawings described below. [Brief description of the drawings]

[0116] [Figure 1]Figure 1 shows heterogeneous sensitivity of AML primary cells to kinase inhibitors. (A) Cell viability of 36 AML cases as a function of ex vivo treatment with the indicated kinase inhibitors at the indicated concentrations. (B) Clustering analysis of AML primary cells based on sensitivity to 1 μM treatment with the indicated compounds. (C) Correlation analysis of sensitivity of AML primary cells to treatment. (D) Sensitivity of AML primary cells to 1 μM MEKi treatment as a function of FAB subtype. p-values ​​were assessed using an unpaired, two-tailed Student's t-test. In (C), the critical values ​​for significant correlation in the two-tailed test were r>0.329 for p<0.05 (*), r>0.428 for p<0.01 (**), and r>0.526 for p<0.001 (***). All experiments were performed on n=36 AML cases.

[0117] [Diagram 2] shows that the phosphoproteome of FAB-M1 and FAB-M4 classifies AML primary cells into groups with specific patterns of protein phosphorylation and kinase activity. (A) Hierarchical cluster analysis of 150 phosphopeptides showing greater significant differences between FAB-M1 and FAB-M4 cases. (B) The overall phosphopeptide profile was significantly increased in the M1-like and M4-like groups. (C) Phosphorylation sites shown in (B) were used to infer activity for the indicated kinases by KSEA. In all experiments, n=36 primary AML samples. In (B), unpaired two-tailed Student's t-test was used to calculate p-values ​​adjusted using the Benjamini-Hochberg procedure (FDR). In (C), the significance of enrichment was assessed using the hypergeometrics test.

[0118] [Diagram 3]shows that surface expression of differentiation markers stratifies AML patients and correlates with specific patterns of protein phosphorylation. (A) Mean surface expression of the indicated differentiation markers for M1-like and M4-like groups. (B) Frequency of correlation between expression of differentiation markers and phosphopeptide abundance. (C-E) Correlation between phosphorylation of MAPK1 at Y187, PAK2 at S141, and PRKCD at Y313 and surface expression of the indicated differentiation markers. (F) Pearson correlation coefficients for expression of each differentiation marker. In all experiments, n=30 primary AML samples. In (A), p values ​​were assessed using unpaired two-tailed Student's t-test. In (F), the critical values ​​for significant correlation in two-tailed tests were r>0.361 for p<0.05 (*), r>0.463 for p<0.01 (**), and r>0.571 for p<0.001 (***).

[0119] [Figure 4] shows that AML cases at different differentiation stages present specific patterns of protein expression, protein phosphorylation, and kinase activity. (A) Hierarchical clustering analysis of 30 AML cases based on the indicated differentiation markers. (B) Summary of protein overexpression across CD groups. (C) Summary of phosphopeptide abundance across CD groups. (D) Gene ontologies associated with proteins differentially phosphorylated across CDs+ and CDs- groups. (E) KSEA estimates of average kinase activity in CDs+ and CDs- groups. In (B) and (C), p-values ​​were calculated using unpaired two-tailed Student's t-test analysis and adjusted using the Benjamini-Hochberg correction. In (D) and (E), p-values ​​were assessed using the hypergeometric distribution test.

[0120] [Diagram 5]Figure 1 shows sensitivity to kinase inhibitors as a function of AML blast differentiation. (A) Viability of primary AML samples classified as M4-like or M1-like (n=36) after 72 h treatment with the indicated inhibitors. (B) Viability of primary AML samples classified as CDs+ or CDs- (n=30) after 72 h treatment with the indicated inhibitors. Statistical significance was assessed using a two-tailed Mann Whitney test.

[0121] [Figure 6] shows that mutations in genes involved in kinase signaling are associated with differentiation stage and specific phosphorylation patterns. (A) Mutations in the indicated genes across analyzed AML primary samples. Kinase sig. represents any activating mutation in either FLT3, NRAS, or BRAF. (B-G) MAPK1 phosphorylation and cell viability after 1 μM treatment with MEKi as a function of the indicated genotype or phenotype. p values ​​were calculated in (B)-(C) and (D)-(E), respectively, using unpaired two-tailed Student's t test and one-way ANOVA followed by Tukey's test.

[0122] [Figure 7]1 shows the integration of genomic, phosphoproteomic, and mass cytometry data to describe kinase inhibitor sensitivity. (A) Viability of AML primary cells following 1 μM treatment with MEKi as a function of the indicated combination of genotype and phenotype. MAPK1 phosphorylation is shown as high (hi) and low (lo) based on having high or low phosphorylation, respectively, compared to the median phosphorylation across all cases. (B) Mann Whitney p-values ​​obtained for the comparisons made in (A). (C) Viability of AML cells with NRAS / BRAF / CDs+ genotype / phenotype and the indicated factors as a function of 1 μM treatment with MEKi. (D) Viability of AML primary cells following 1 μM treatment with MEKi as a function of the indicated combination of genotype and phenotype. (E) Mann Whitney p-values ​​for viability following treatment of AML cells with 1 μM of the indicated inhibitor as a function of the presence / absence of the indicated genotype / phenotype. (F) Survival of AML cells with the indicated phenotypes / genotypes as a function of FLT3 / PKCi treatment. Statistical significance in (C-D, F) was assessed using a two-tailed Mann Whitney test. Where p-value numbers are not presented, *p<0.05; **p<0.01; ***p<0.001.

[0123] [Figure 8] Figure 1 shows dose-response curves for cell viability of primary AML cells treated with a panel of kinase inhibitors. (A) Dose-response curves to kinase inhibitors for each AML primary sample. (B) Dose-response curves to kinase inhibitors for AML primary samples clustered based on FAB subtype. Data points are mean ± SD (n=3). Patient biopsies were obtained from the Barts Cancer Institute biobank with ethical consent.

[0124] [Figure 9]shows that peptide phosphorylation-based differentiation signatures stratify AML patients into groups with distinct patterns of phosphorylation. (A) Heatmap showing phosphorylation of 150 peptides used to define M4-like and M1-like groups across 36 primary AML samples sorted based on FAB classification clusters. (B) Samples classified as M4-like hyperphosphorylate membrane and cytoplasmic proteins that favor GTPase signaling, whereas M1-like samples hyperphosphorylate nuclear proteins with DNA- and RNA-binding properties. p-values ​​were assessed using the hypergeometric distribution test.

[0125] [Figure 10] Figure 1 shows AML primary cells classified by differentiation phosphoproteomic signatures in groups displaying defined patterns of kinase phosphorylation and surface expression of differentiation markers. M1-like and M4-like groups were defined using hierarchical clustering based on 150 phosphopeptides that were differentially expressed between AML cases classified as M1 or M4 FAB subtypes. (A) Phosphorylation sites in kinases (n=36) that were significantly regulated (p-value <0.05) between M1-like and M4-like groups. (B) List of surface markers quantified by mass cytometry. (C) Differentially expressed differentiation markers (n=30) between M1-like and M4-like groups. p-values ​​were assessed using unpaired two-tailed Student's t-test.

[0126] [Figure 11] shows the significance of the association between surface expression of phospho- and CD markers across 30 AML cases.

[0127] [Figure 12]A shows differential protein expression and differentially phosphorylated kinases in the CDs+ and CDs- groups. Heatmap showing peptides contained in proteins (A) or kinases (B) whose expression or phosphorylation was significantly increased (p-value < 0.05) in either of the CDs groups. p-values ​​were evaluated using an unpaired two-tailed Student's t-test.

[0128] [Figure 13] Figure 1 shows dose-response curves to kinase inhibitors for AML primary samples clustered into the CDs group. Survival of primary AML samples classified as CDs+ (n=12) or CDs- (n=18) after 72 h treatment with the indicated inhibitors. p-values ​​were assessed using an unpaired two-tailed Student's t-test: *p<0.05; **p<0.01; ***p<0.001.

[0129] [Figure 14] Figure 1 shows the viability of AML cells with the indicated genotype / phenotype as a function of MEKi treatment. (A) Viability of AML primary cells after treatment with MEKi. (B) Viability of AML cells after treatment with FLT3 / PKCi. (C) Viability of AML primary cells after treatment with CK2i. p values ​​were assessed using a two-tailed Mann Whitney test. *p<0.05; **p<0.01; ***p<0.001. EXAMPLES

[0130] Research Overview This study was performed on 36 randomly selected primary samples of mononuclear cells extracted from peripheral blood of AML patients at the time of diagnosis. Experiments were performed as described below to determine the in vitro viability of cells in response to treatment with inhibitors of the kinases FLT3 / PKC (midostaurin, FLT3 / PKCi), PAK (PF-3758309 PAKi), CK2 (silmitasertib CK2i), and MEK (trametinib, MEKi). The P38 inhibitor (P38i) TAK-715 was included as a negative control. Cells obtained from 36 AML patients with well-annotated clinical data were treated with these compounds for 72 hours.

[0131] Dose-response curves showed, as expected, that the 36 tested samples showed heterogeneous responses to all compounds (FIGS. 1A-B, and FIG. 8A). Because it is difficult to interpret drug response curves if treatment does not reduce viability by 50% or more, we used a 1 μM dose (expected to inhibit the kinase of interest based on the compound's reported in vitro IC50) to define sensitivity to treatment. At the 1 μM dose, PAKi was the most potent of all compounds tested, reducing survival by >50% in 18 / 36 (50%) AML cases compared with DMSO controls, followed by FLT3 / PKCi (9 / 36, 25%), MEKi (8 / 36, 22%), and CK2i (8 / 36, 22%). At the same threshold, P38i treatment alone reduced survival in three AML cases (8%).

[0132] Clustering analysis of the cell sensitivity data showed that PAKi-sensitive cells also tended to be sensitive to MEKi and FLT3 / PKCi (Fig. 1B-C). In contrast, the response rates to MEKi, PAKi, or FLT3 / PKCi were very different from those to CK2i and P38i, suggesting that PAKi, MEKi, and FLAT3 / PKCi have very similar, though not identical, mechanisms of action that are different from those of CK2i and P38i.

[0133] Example 1 More differentiated AML cells are more responsive to kinase inhibitor treatment than less differentiated cells. We found that M4 cells were significantly more responsive to MEKi than M1 cells (Figure 1D), suggesting that AML cases of M4 subtype showed higher sensitivity to MEKi than those classified as M1, indicating that the differentiation state of leukemic cells may be a marker of sensitivity to kinase inhibitor therapy.

[0134] Example 2 Identifying phosphoproteomic signatures characteristic of differentiated cells We investigated differences in kinase signaling between these AML subtypes. Using mass spectrometry methods described below, we identified and quantified 9,534 phosphopeptide ions in these experiments. Of these, we selected 150 phosphorylation sites showing the most significant differences (based on p-values ​​of Student's t-test) between the groups as a phosphoproteomic signature distinguishing M4 from M1 AML subtypes (Figure 9A). Since M4 cells are more differentiated compared to M1, we hypothesized that this signature may be related to the differentiation stage of the analyzed blasts. In hierarchical clustering analysis, this phosphoproteomic signature subdivided our cohort of 36 patients into two defined groups (Figure 2A): we named the group containing 10 of the 12 cases of M1 subtype "M1-like" and the group containing all M4 cases "M4-like" (Figures 2A and 2B). The M1-like and M4-like groups consisted of 16 and 20 cases, respectively.

[0135] We used ontology enrichment analysis and kinase substrate enrichment analysis (KSEA) to explore the biological processes and signaling pathways enriched in the various groups. Analysis of phosphoproteomic differences between cases ( Fig. 2B ) showed that M4-like cases had an increase in cytoplasmic and membrane phosphoproteins involved in GTPase signaling, whereas M1-like cases had an increase in nuclear phosphoproteins with DNA- and RNA-binding properties. KSEA, a computational procedure that estimates individual kinase activity based on the phosphorylation of known substrates, showed that activities of PKC, ERK, PAK1, and P38α were enriched in the M4-like group of cells, whereas activities of CDK7, CK1A, and AurB were enriched in M1-like cells (Figure 2C). Several of the increased phosphorylation sites in the M4-like group were present in kinases at regulatory sites (eg, PAK1 at S144, PAK2 at S141, MAPK1 at Y187, and RPS6KA1 at S380) (FIG. 10A). These data indicate that M4-like cells activate kinase signaling pathways known to act downstream of cell surface receptors (e.g., PKC, MAPK, and PAK kinases) to a greater extent than M1-like cells.

[0136] Example 3 Identification of cell surface marker protein signatures that are characteristic of differentiated cells and correlate with phosphoproteomic signatures To precisely measure differentiation status, we used mass cytometry to immunophenotype 30 cases from a cohort of 36 AML for which we had material available, by measuring surface expression of 17 differentiation markers (Figure 10B). We found that M4-like cases had higher expression of myelomonocytic differentiation markers compared with M1-like cases (Figures 3A and 10C). Next, we investigated whether the presence of specific differentiation markers was associated with the activation of kinase signaling pathways. We found that surface expression of CD45, CD11b, CD44, CD14, CD16, CD64, and CD15 was statistically associated (r>0.7, p<0.001) with phosphorylation patterns at 80–219 sites per marker (Figure 3B). Examples include phosphorylation of ERK2 (MAPK1 gene) at Y187, phosphorylation of PAK2 at S141, and phosphorylation of PKCδ (gene PRKCD) at Y313, which were statistically associated with the expression of several differentiation markers (scatter plots for MAPK1 phosphorylation sites, PAK2 phosphorylation sites, and PKCδ phosphorylation sites are shown in Figure 3C-E, and the associated p-values ​​are shown in Figure 11). We found that CD markers were co-expressed (Fig. 3F). Hierarchical clustering subdivided our cohort of 30 patients into two groups, which we termed CDs+ and CDs- (Figure 4A), which consisted of 12 and 18 cases, respectively, and overlapped with the M4-like and M1-like groups. In this and the following examples, we focus on CDs + Cells were characterized for surface expression of a panel of cell surface markers consisting of CD33, CD123, HLA-DR, CD44, CD38, CD15, CD45, CD16, CD64, CD11b, and CD14.

[0137] Example 4 Identifying protein marker signatures characteristic of differentiated cells To examine in more detail the biochemical differences in AML blasts as a function of the differentiation state of the cells, we compared differences in proteomic, phosphoproteomic, and kinase activities between CDs+ and CDs- AML cases. This proteomic analysis identified 2,391 proteins (Figure 4B), revealing a set of proteins (already associated with differentiation) that showed higher expression in the CDs+ population compared to CDs-, including integrins, lysozyme C, and other proteins associated with myeloid differentiation (Figure S12A). Interestingly, several kinases, phosphatases, and signaling regulators were also expressed at higher levels in CDs+ compared to CDs- cases (Figure 12A).

[0138] Regarding the results of phosphoproteomic analysis, CDs+ cases showed an approximately three-fold increase in phosphorylation sites compared with CDs- cases (Figure 4C). Ontology enrichment analysis highlighted the expression of phosphoproteins associated with immune, GTPase, and kinase signaling in CSs+, while CD− cases showed increased abundance of nuclear phosphoproteins and those associated with the regulation of transcription (Figure 4D). Kinases with increased phosphorylation in the CDs+ group compared with CDs− cases included FES at Y713, ERK1 (MAPK3) at T202 / Y204, ERK2 (MAPK1) at T185 / Y187, PAK1 at S144, MEK1 (MAP2K1) at S222, PAK2 at S141, and PKC-δ (PRKCD) at S645 ( Figure 12B ). Consistent with these observations, the CDs+ group had enhanced activity of several kinases (e.g., PKA, several isoforms of PKC, BRAF, MEK, and ERK) compared with CDs- cells (Figure 4E). The increased expression of integrins, survival kinases, and other signaling regulators in CDs+ cells compared with CDs- cells (Figure S12A), suggesting that the increased activation of kinase pathways in more differentiated cells (Figure 4E) is at least in part due to higher expression of these signaling molecules.

[0139] Example 5 M4-like and CDs+ cells are highly sensitive to kinase inhibitor therapy Because M4-like and CDs+ cases activated kinase survival pathways to a greater extent than M1-like and CDs- cases, respectively, we hypothesized that there may be differences in how cells from these patient groups may respond to kinase inhibitors. Consistently, analysis of cell viability as a function of treatment with kinase inhibitors showed that M4-like and CDs+ cases displayed greater sensitivity to 1 μM PAKi, 1 μM MEKi, and 10 μM FLT3 / PKCi, concentrations that can inhibit PKCδ, a kinase that was found to be highly active in our assays (Figures 2C, 3E, 4E), compared to M1-like and CDs- (Figures 5A-B). The same trend in response was observed after treatment with other compound concentrations (Figure 13). There were no differences in response to CK2i or P38i between groups ( Fig. 5A-B ). Collectively, phosphoproteomics and differentiation marker expression stratified AML patients into groups with strikingly different patterns of kinase activity and sensitivity to FLT3 / PKC, PAK, and MEK inhibitors. Thus, these results suggest a link between differentiation, activity of kinase-driven survival pathways, and the sensitivity of AML cells to kinase inhibitors.

[0140] Example 6 Mutational status as a marker of kinase inhibitor sensitivity To investigate mechanisms that may contribute to the pharmacological and biochemical differences observed in AML with distinct differentiation phenotypes, we sequenced the 25 most frequently mutated genes in AML in our sample cohort. We found that 15 of these genes were mutated in at least 1 of the 27 cases included in this analysis (Figure 6A).Interestingly, genes with roles in kinase signaling (e.g., NRAS, BRAF, and FLT3) were more frequently mutated in cells from the CDs+ group (Figure 6A, p=0.008 by hypergeometric distribution test).

[0141] We found that cells with NRAS or BRAF mutations increased phosphorylation of MAPK1 (ERK2) at T185 and Y187, markers of RAS / MEK / ERK pathway activity (Figure 6B). Consistent with published studies, we found that mutations on these genes were also significantly associated with cellular sensitivity to MEKi (Figure 6C). Cells with FLT3 mutations (in a NRAS / BRAF WT background) also displayed relatively high RAS / MEK / ERK pathway activity (as assessed by MAPK1 phosphorylation; Fig. 6D ), but did not display increased sensitivity to MEKi compared with cells WT for FTL3, NRAS, and BRAF ( Fig. 6E ), suggesting that the background of genes associated with pathway activation influences the response to pathway inhibition. Also interestingly, CDs+ cases negative for NRAS / BRAF mutations showed higher RAS / MEK / ERK pathway activation compared to CDs- samples (Figure 6F) and showed higher sensitivity to MEKi compared to anaplastic cases of the same NRAS / BRAF genotype (Figure 6G).

[0142] Example 7 Mutational and differentiation status as combined markers of kinase inhibitor sensitivity To further rationalize the response, we performed a systematic analysis integrating the mutational profiles of cells with mass spectrometry and mass cytometry data. Cells with mutated NRAS, high MAPK1 phosphorylation, or positivity for CDs+ were more sensitive to MEKi than cells WT for NRAS, low for MAPK1 phosphorylation, or negative for the CDs phenotype, respectively (Figure 7A(i-iv)). Cells with the NRAS / BRAF / FLT3-ITD genotype did not show higher sensitivity to MEKi compared with cells with either NRAS or BRAF mutations alone (Figure 7A(v)). In contrast, the 15 cases positive for either NRAS / BRAF / CDs+ showed, on average, higher sensitivity to MEKi compared with cells lacking this molecular signature (Figure 7A(vi-ix)).

[0143] To assess the significance of the differences in MEKi sensitivity as a function of the various molecular markers, we plotted the p-values ​​of the comparisons shown in Figure 7A. Combining the NRAS / BRAF / CDs+ signature yielded a log 10 The most significant differences occurred with p-values ​​and log 10 This was followed by the NRAS / BRAF / MAPK1hi / CDs+ signature with a p-value of -5.0 (Figure 7B). These results suggest that AML cells may activate the MEK / ERK pathway either through mutations on NRAS / BRAF or surface expression of CD markers, which results in the AML cells being more sensitive to MEKi treatment compared to cells with WT genes or less differentiated states.

[0144] Cases with either NRAS / BRAF mutations or a CDs+ phenotype (NRAS / BRAF / CDs+ cases) showed high sensitivity to MEKi, with 8 of 15 cases with this signature showing >50% survival after treatment (Figure 7(viii)). To investigate the reasons for these differences in response within NRAS / BRAF / CDs+ cases, we compared the mutation status and phosphoproteome in 15 cases positive for NRAS / BRAF / CDs+. Among these 15 cases, we found that FLT3-ITD-positive cells were significantly more resistant to MEKi than cells without this mutation (p=0.012, FIG. 7C). We also found that several phosphorylation markers (e.g., at STAT5A S780, STAT5A S128, TOP2A S1213, KDM5C S317, and CAMKK1 S458) were associated with response to MEKi within NRAS / BRAF / CDs+ cases (Figure 7C), suggesting that cells with the NRAS / BRAF / CDs+ signature but relatively resistant to MEKi may rely on FLT3-driven pathways (e.g., STAT5 29 ) is suggested. Accordingly, samples that were positive for NRAS / BRAF / CDs+ and negative for FLT3-ITD or had low STAT5A or KDM5C phosphorylation were more sensitive to 1 μM MEKi than other cells (Figure 7D), and essentially all NRAS / BRAF / CDs+ cases with low KDM5C phosphorylation were sensitive to MEKi treatment (survival <50%) (Figure 7D, middle panel). In addition, we found that NRAS / BRAF / CDs+ cases negative for FLT3-ITD or with low KDM5C phosphorylation were more sensitive to other concentrations of MEKi, and this difference was greater than when considering NRAS / BRAF in CDs status alone (Figure 14A).

[0145] To identify determinants of sensitivity to inhibitors other than MEKi, we compared sensitivity to compounds as a function of mutational status, expression of phosphorylation markers, or a combination of the two (Figure 7E). We found that NRAS mutations were the only strong genomic determinant of sensitivity to MEKi, while IDH2 mutations showed a small (p 0.03) effect on response to CK2i (Figure 7E, left panel). Surprisingly, FLT3-ITD status had no effect on cellular responses to FLT3 / PKCi (Fig. 7F and Fig. S14B). In contrast, we found that several phosphorylation markers, such as those on protein kinase C isoforms (gene names PRKCA, PRKCB, and PRKCD), STK10, GSK3A, and PAK1 / 2, and on the platelet membrane receptor Gi24 (C10orf54), were associated with responses to PAKi, FLT3 / PKCi, and MEKi (Fig. 7E, middle panel). Integration of genomic or CDs markers with phosphorylation data increased the significance of the associations (decreased p-values) (Figure 7, right panel). For example, samples positive for either CDs or phosphorylation on GSK3A or PCKδ showed higher sensitivity to FLT3 / PKCi compared to other cases (Figure 7F and Figure S14B). Regarding CK2i, there was a small association between CD34 expression or IDH2 mutations and sensitivity to this compound, but this effect was modest (FIG. 14C). Taken together, our results suggest that integration of differentiation status (defined by expression of CD markers) with genomic and phosphoproteomic signatures gives rise to groups of AML cases characterized by degrees of sensitivity to MEKi and FLT3 / PKCi.

[0146] Example 8 A companion diagnostic test to assess suitability of AML patients for treatment with midostaurin Peripheral blood or bone marrow samples were obtained from patients suffering from acute myeloid leukemia. Mononuclear leukemia cells were extracted from these samples and assays were performed on the cells as described herein to detect: (i) surface expression of the group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; and / or (ii) high levels of phosphorylation in the patient's leukemia cells at any one or more of pS21 of GSK3A; pY313, pT507, pT295, pT218, and / or pS664 of PRKCD; pS20 and / or pS13 of STK10; pS144 of PAK1; pS141 of PAK2; Y187 and / or T185 of MAPK1; and T202 and / or Y204 of MAPK3. In step (ii), a high level of phosphorylation was identified if phosphorylation at the reference site was higher than the average phosphorylation at that site calculated across multiple patient samples. Patients whose cells were positive for either (i) or (ii) were identified as suitable for treatment with midostaurin.

[0147] Example 9 A companion diagnostic test to assess suitability of AML patients for treatment with trametinib Peripheral blood or bone marrow samples were obtained from patients suffering from acute myeloid leukemia. Mononuclear leukemia cells were extracted from these samples and assays were performed on the cells as described herein to detect: (a)(i) surface expression on the patient's leukemic cells of the group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; and / or (ii) high levels of phosphorylation in the patient's leukemic cells at any one or more of Y187 and / or T185 of MAPK1; and T202 and / or Y204 of MAPK3; (iii) an activating mutation in any one or more of NRAS, KRAS, HRAS, or BRAF in the patient's leukemia cells; and (b)(i) high levels of phosphorylation at one or more of pS780 and pS128 of STAT5A, pS548 of CAMKK1, pS1213 of TOP2A, and pS317 of KDM5C; and / or (ii) the absence of any activating mutations in FLT3 in this patient's leukemic cells. wherein in steps (a)(ii) and (b)(i), a high level of phosphorylation was identified if phosphorylation at a reference site was higher than the average phosphorylation at that site calculated across multiple patient samples. Patients whose cells were positive for both (a) and (b) were identified as suitable for treatment with trametinib.

[0148] Consideration A central goal of targeted therapy is to identify actionable patient-specific pathways that can direct effective individualized treatment. In this study, we discovered that across AML samples, differentiation state determines the extent and / or nature of kinase pathway activation. Some of the surface differentiation markers (eg, CD45 and CD123) are membrane receptors or play a role in the recognition of extracellular signals that are transduced and propagated intracellularly by protein kinase cascades. Cells positive for these CD markers showed higher expression of proteins associated with myelomonocytic differentiation and kinase signaling compared to CDs- cells, and consequently increased phosphorylation and activation of pro-survival kinases (Figures 2-4), explaining the increased sensitivity in how these cells responded to treatment with the PAKis, midostaurin, and trametinib (Figure 5).

[0149] By integrating mass spectrometry and cytometry data with recurrent mutations, AML is more predictive than other studies. 14 Consistent with this, we showed that activating mutations in NRAS were associated with higher ERK (MAPK) activity and conferred sensitivity to MEKi (Figures 6C-D). In our cohort of patients, NRAS mutations, when considered in isolation, appeared to be the only clear genomic determinant of response, and surprisingly, neither FLT3-ITD nor FLT3-TKD mutations were associated with response to midostaurin (Figure 7F). Our data suggest that the RAS / MEK / ERK pathway may be activated in AML by the presence of NRAS / BRAF activating mutations or by signals originating from upstream cell surface CD markers or related receptors. Therefore, MEKi treatment was likely to reduce the viability of AML cells that were positive for at least one of these markers (Fig. 7B).

[0150] However, despite the clear contribution of RAS / MEK / ERK activation to the magnitude of response to MEKi, only approximately 50% (7 / 15) of cases positive for RAS / MEK / ERK activation showed a high response to MEKi. We found that cases that were relatively resistant to MEKi had the FLT3-ITD genotype and high levels of phosphorylated regulatory proteins (e.g., STAT5A, KDM5C, and tomoisomerase 2A at S1213, a site that regulates the activity of the enzyme) despite activation of the RAS / MEK / ERK pathway (Figure 7). Thus, AML cell populations that respond well to MEKi exhibit high activity of their target pathways (RAS / MEK / ERK) along with low activity of the FLT3 / STAT pathway (Fig. 7D), which is known to maintain the viability and proliferation of primary AML cells by acting in parallel with RAS / MEK / ERK signaling.

[0151] Thus, our results suggest two distinct mechanisms of intrinsic resistance to MEK inhibition. The first occurs in cells that are not dependent on the pro-survival effects of MEK because these cells have low RAS / MEK / ERK pathway activity. The second mechanism occurs in cells that have highly active RAS / MEK / ERK but bypass MEK inhibition because the FLT3 / STAT5 axis acts as a compensatory mechanism.

[0152] Pemovska et al. observed that 36% of samples from AML patients showed increased sensitivity to trametinib compared to mononuclear cells from healthy donors, and we found that 22% of our cases showed a greater than 50% decrease in survival as a result of treatment with this drug (Figure 1). Therefore, it is worth considering repurposing trametinib, a drug already approved by the FDA to treat melanoma, to treat the 20-35% of AML cases expected to respond to such treatment.

[0153] Regarding PAKi and FLT3 / PKCi, we found no association between genetic alterations and response to these compounds (FIG. 7). However, we observed that cells with a more differentiated phenotype and with high phosphorylation of GSK3A and PKCδ responded better to midostaurin compared to cells with low phosphorylation on these markers (note that PKCδ is upstream of GSK3A [Ref 38 ]). Therefore, our results support clinical development. 39 , 4 0 These results suggest that the mechanism of action of midostaurin (the FLT3 / PKCi trialed in this study) involves inhibition of PKCδ, a known target of this drug.

[0154] In conclusion, we discovered that AML cells activate a receptor tyrosine kinase signaling network during differentiation, resulting in a marked increase in the activity of pro-survival pathways regulated by MEK and PKC isoforms. The combination of targeted and parallel kinase pathway activation (driven by genetic and non-genetic events) determined the extent to which AML cells responded to treatment with trametinib or midostaurin.

[0155] method Study design The study was performed on 36 primary samples of mononuclear cells extracted from peripheral blood of AML patients at the time of diagnosis. Samples were randomly selected from the BCI tissue bank collection. Initially, the study included 45 samples, but 9 were later excluded because they were not viable in ex vivo experiments. The availability of material allowed proteomic and mass cytometric analysis of 30 samples and DNA sequencing of 27. Ex vivo drug testing was performed in quadruplicate sampling replicates and viability values ​​were averaged and expressed relative to vehicle control.

[0156] The patient gave informed consent for the storage and use of her blood cells for research purposes. Experiments were performed in accordance with the Local Research Ethics Committee.

[0157] mass spectrometry Cells were lysed and proteins digested using trypsin as previously described in Wilkes EH, Terfve C, Gribben JG, Saez-Rodriguez J, Cutillas PR. Empirical inference of circuitry and plasticity in a kinase signaling network. Proc Natl Acad Sci US A. 2015;112(25):7719-7724.

[0158] LC-MS / MS identification and quantification of peptides and phosphopeptides were performed on an orbitrap mass spectrometer (Q-Exactive Plus). Normalized quantitative data were used to calculate fold changes between groups and statistical significance (assessed by Student's t-test). Mass spectrometry proteomic data have been deposited with the ProteomeXchange Consortium via the PRIDE partner repository with dataset identifier PXD005978 and DOI 10.6019 / PXD005978. See Vizcaino JA, Csordas A, Del-Toro N et al., 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 2016;44(22):11033.

[0159] Bioinformatics Estimation of kinase activity from phosphoproteomic data was performed using kinase substrate enrichment analysis (KSEA) as described in Casado P, Rodriguez-Prados JC, Cosulich SC, et al. Kinase-substrate enrichment analysis provides insights into the heterogeneity of signaling pathway activation in leukemia cells. Sci Signal. 2013;6(268):rs6.

[0160] Gene ontology (GO) enrichment was determined using DAVID software ( https: / / david.ncifcrf.gov / ), and the gene ontology was considered enriched if the Bonferroni corrected p value was <0.05. Hierarchical clusters were constructed within the R statistical computing environment (3.2.3) using the Euclidean distance metric with the Heatmap2 function.

[0161] Mass Cytometry Primary cells were coated with metal-conjugated antibodies and analyzed on a CyTOF2 mass cytometer (Fluidigm) as instructed by the manufacturer. Data were normalized using the normalizer within the DVS Sciences CyTOF Instrument Control Software (v6.0.626).

[0162] Viability assay Ex vivo drug testing in primary AML cells was as previously described in Casado P, Rodriguez-Prados JC, Cosulich SC, et al. Kinase-substrate enrichment analysis provides insights into the heterogeneity of signaling pathway activation in leukemia cells. Sci Signal. 2013;6(268):rs6. Briefly, cells were resuspended in MS-5 conditioned IMDM medium, plated in 96-well plates, and treated with vehicle or the indicated inhibitors at 1-10,000 nM for 72 h. Cells were stained with Guava ViaCount reagent to determine cell number and viability. Flow cytometry data were analyzed using CytoSoft (v2.5.7).

[0163] Panel sequencing Target enrichment of a 25-gene bone marrow panel was achieved using an in-house True SeqCustom Amplicon (TSCA) design (Illumina, San Diego, USA).

[0164] statistical analysis Statistical analyses were performed in R (version 3.2.3), Micorsoft Excel 2013, or Prism (version 5.4). p values ​​returned from Mann Witney, Anova, or Student's t tests (shown in figures) were adjusted for multiple testing using Tukey or Benjamini-Hochberg procedures, as appropriate.

[0165] Mass spectrometry, proteomics, and phosphoproteomics Cells were harvested by centrifugation at 500×g for 5 min at 4° C., washed twice with cold PBS supplemented with 1 mM Na 3 VO 4 and 1 mM NaF, snap frozen, and stored at −80° C. until further processing. The cell pellet was lysed in urea buffer (8 M urea in 20 mM HEPES pH 8.0 supplemented with 1 mM Na3VO4, 1 mM NaF, 1 mM Na4P2O7, and 1 mM sodium β-glycerophosphate) for 30 min and further homogenized by sonication (60 cycles of 30 sec on, 40 sec off; Diagenode Bioruptor® Plus, Liege, Belgium). Insoluble material was removed by centrifugation at 20,000×g for 10 min at 5° C., and protein in the cell extracts was quantified by bicinchoninic acid (BCA) assay.

[0166] For phosphoproteome analysis we used published methods with some modifications, see below: Gruhler A,Olsen JV,Mohammed S,Mortensen P,Faergeman NJ,Mann M,et al.Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Molecular & cellular proteomics:MCP 2005;4(3):310-27 doi 10.1074 / mcp.M400219-MCP200. Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jorgensen TJ. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.Molecular & cellular proteomics:MCP 2005;4(7):873-86 doi 10.1074 / mcp.T500007-MCP200. Montoya A, Beltran L, Casado P, Rodriguez-Prados JC, Cutillas PR.Characterization of a TiO(2)enrichment method for label-free quantitative phosphoproteomics.Methods 2011;54(4):370-8 doi 10.1016 / j.ymeth.2011.02.004

[0167] Briefly, 250 μg of protein was reduced and alkylated by sequential incubation with 10 mM DTT and 16.6 mM iodoacetamyde for 1 h. The urea concentration was diluted to 2M with 20 mM HEPE, pH 8.0, and 80 μL of conditioned trypsin beads (conditioned by three washes with 20 mM HEPES, pH 8.0 [50% slurry of TLCK-trypsin (Thermo-Fisher Scientific; Cat. #20230)]) was added and the sample was incubated with agitation at 37° C. for 16 hours. Trypsin beads were removed by centrifugation at 2,000×g for 5 min at 5° C. For phosphoproteomic analysis, 100 μg of protein was used.

[0168] After trypsin digestion, the peptide solution was desalted using 10 mg OASIS-HLB cartridges (Waters, Manchester, UK). Briefly, OASIS cartridges were placed in a vacuum manifold (-5 mmHg), activated with 1 mL ACN, and equilibrated with 1.5 mL wash solution (1% ACN, 0.1% TFA). After loading the sample, the cartridge was washed with 1 mL of wash solution. For phosphoproteomic analysis, it was eluted with 500 μL of glycolate buffer 1 (1 M glycolic acid, 50% ACN, 5% TFA) and subjected to phosphoenrichment. For proteomic analysis, peptides were eluted with 500 μL of ACN solution (30% ACN, 0.1% TFA), dried in a speed vac (RVC 2-25, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) and stored at −80 °C.

[0169] Phosphopeptides were enriched using TiO2 (GL Sciences) as previously described (4) with some modifications: Sample volume was normalized to 1 mL using glycolic acid buffer 2 (1 M glycolic acid, 80% ACN, 5% TFA), and 50 μL of TiO2 beads (50% slurry in 1% TFA) was added to the peptide mixture, incubated with stirring for 5 min at room temperature, and centrifuged at 1500 × g for 30 s. For each sample, 80% of the supernatant was transferred to a fresh tube and stored on ice, and the remaining 20% ​​was used to resuspend the bead pellet loaded into an empty pre-washed PE-filtered spin tip (Glygen, MD, USA) and combined by centrifugation at 1500 × g for 3 min. After loading the remaining volume of the supernatant by centrifugation at 1500×g for 3 min, the spin chip was washed sequentially with 100 μL of glycolate buffer 2, ammonium acetate buffer (100 mM ammonium acetate in 25% ACN), 10% ACN by centrifugation at 1500×g for 3 min at RT. For recovery of phosphopeptides, the addition of 50 μL of 5% aqueous ammonia followed by centrifugation at 1500×g for 5 min was repeated four times, the eluate was snap frozen in dry ice, dried in a speed vac, and the peptide pellet was stored at −80° C.

[0170] For phosphoproteomics, peptide pellets were resuspended in 12 μL of reconstitution buffer (20 fmol / μL enolase in 3% ACN, 0.1% TFA) and 5.0 μL was loaded onto an LC-MS / MS system consisting of a Dionex UltiMate 3000 RSLC directly connected to an Orbitrap Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific). For proteomics, the pellet was resuspended in reconstitution buffer (0.5 μg / μL) and 2 μL was injected. The LC system used mobile phases A (3% ACN; 0.1% FA) and B (100% ACN; 0.1% FA). Peptides were captured on a μ-pre-column (cat. no. 160454) and separated on an analytical column (Acclaim PepMap 100; cat. no. 164569). The following parameters were used: a gradient from 3% to 23% B over 120 min, and a flow rate of 0.3 μL / min.

[0171] Upon elution from the nano-LC system, peptides were injected into an on-line connected Q-Exactive Plus system operating at a duty cycle of 2.1 seconds. Acquisition of a full-scan survey spectrum (m / z 375-1,500) with 70,000 FWHM resolution was followed by a data-dependent acquisition in which the 20 most prominent ions were selected for HCD (higher-energy collisional dissociation) and MS / MS scanning (200-2,000 m / z) with a resolution of 17,500 FWHM. A dynamic exclusion period of 30 seconds was allowed by an exclusion list with a mass window of 10 ppm. The overall duty cycle yielded a chromatographic peak of approximately 30 seconds on base, which allowed the construction of an extracted ion chromatogram (XIC) with at least 10 data points. I also uploaded the raw files of the extra samples to PRIDE.

[0172] Peptide identification and quantification Peptide identification from the MS data was automated using Mascot Daemon 2.5.0. Peak list files (MGF) from the raw data were generated with Mascot Distiller v2.5.1.0 and loaded into the Mascot search engine (v2.5) to match MS / MS data to peptides (Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999;20(18):3551-67). Searches were performed against the SwissProt Database (SwissProt_2012Oct.fasta for proteomics, or uniprot_sprot_2014_08.fasta for phosphoproteomics analysis) with an FDR of approximately 1% and the following parameters: two tryptic deletion cleavages, a mass tolerance of ±10 ppm for MS scans and ±25 mmu for MS / MS scans, carbamidomethyl Cys as fixed modification, PyroGlu on the N-terminal Gln and oxidation of Met as variable modifications. For phosphoproteomics experiments, phosphorylation of Ser, Thr, and Tyr were also included as variable modifications. Pescal software developed in-house was used for label-free peptide quantification (6), and XICs for all peptides identified across all samples were constructed with mass and retention time windows of ±7 ppm and ±2 min, respectively. Peak areas from all XICs were calculated. Undetectable peptides were assigned an intensity value of 0. Values ​​from two technical replicates per sample were averaged, and the intensity value of each peptide was normalized to the total sample intensity.

[0173] Mass Cytometry Mass cytometry was used to characterize CD markers in AML cells (Bandura DR, Baranov I, Ornatsky OI, Antonov A, Kinach R, Lou X, et al. Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Analytical chemistry 2009;81(16):6813-22). Cells (4 × 10 6 ) were transferred to a fresh tube, washed twice with PBS and incubated with 1× Cell-ID™ Cisplatin solution (Fluidigm; Cat. 201064) for 5 min at RT. Cells were washed with Maxpar Cell Staining buffer, the pellet resuspended and incubated with 50 μL of 20 μg / mL HAG (human gamma-globulin, Sigma-Aldrich; Cat. G4386-1G) for 20 min at RT. After addition of 50 μL of antibody mix (1 / 50 dilution of each antibody; Supplementary Table 1), samples were incubated at RT for 30 min. Cells were then washed twice with Maxpar Cell Staining buffer and the pellet was resuspended in Fix and Perm Buffer and left overnight at 4° C. The next day, Ir intercalator was added to a final concentration of 1× and samples were incubated for 20 min at RT. Permeabilized cells were washed twice with Maxpar Cell Staining buffer and then twice with Maxpar water.

[0174] The following antibodies were used in the mass cytometry assays, as described below: (Table 3) Antigen Determinant Clonal Metal CD19 CD21 coreceptor HIB19 142Nd CD117 receptor tyrosine kinase / SCF 104D2 143Nd CD11b Fibrinogen Receptor ICRF44 144Nd CD64 Fc receptor 10.1 146Nd CD7 CD7-6B7 147Sm CD123 Interleukin 3 receptor 6H6 151Eu CD45 receptor tyrosine phosphatase HI30 154Sm CD33 Sialic acid receptor WM53 158Gd CD15 Carbohydrate W6D3 164Dy CD34 Intercellular adhesion factor 581 166Er CD3 TCR coreceptor UCHT1 170Er CD44 Hyaluronan Receptor IM7 171Yb CD38 Cyclic ADP synthesis HIT2 172Yb HLA-DR antigen presentation L243 174Yb CD184 Chemokine receptor / PSD-1 12G5 175Lu CD14 TLR4 coactivator M5E2 160Gd CD16 Fc receptor 3G8 148Nd

[0175] Sanger sequencing Primers for BRAF V600 PCR were forward 5′-TCTTCATGAAGACCTCACAGT-3′ and reverse 5′-CCAGACAACTGTTCAAACTGA-3′. 20-50 ng of DNA was used as template, and the thermal conditions were as follows: An initial heating period of 15 minutes at 95°C; 36 cycles at 95° C. for 1 min, 55° C. for 1 min, and 72° C. for 1 min, and Final 10 min at 72°C. Amplification products were sequenced by GATC Biotech (Constanza, Germany) using the forward primer. Positive cases were verified using the reverse primer.

[0176] Mass Cytometry Using mass cytometry, we simultaneously measured the surface expression of 17 differentiation markers (CD3, CD7, CD11b, CD14, CD15, CD16, CD19, CD33, CD34, CD38, CD44, CD45, CD64, CD117, CD123, CD184, and HLA-DR) in mononuclear cells extracted from peripheral blood of 30 patients with AML. Label-free quantitative phosphoproteomics based on TiO2 phosphoenrichment was used to quantitate over 5,000 phosphorylation sites in the same primary AML samples, and KSEA technology was applied to infer kinase activity from phosphoproteomic data. Gene ontology enrichment was calculated based on protein-coding genes for which phosphorylation sites were detected using DAVID software. Guava EasyCyte Flow Cytometry was used to determine cell viability following treatment of the same patient samples with five kinase inhibitors (PF-3758309, midostaurin, silmitasertib, trametinib and TAK715, which aim to target the kinases PAK, Flt-3, casein kinase 2, MEK and P38, respectively).

Claims

1. 1. A method for predicting the efficacy of a kinase pathway inhibitor in treating acute myeloid leukemia in an individual patient, the kinase pathway inhibitor inhibiting a signaling pathway involved in cell proliferation or cell survival, the method comprising the steps of: (a) determining the differentiation state of leukemic cells of said patient by: (1) FES, PKS, and protein kinase C isoforms, PKA, PAK, STK10, GSK3A, RSK2, RAS, RAF, MEK, ERK, PI3K, AKT, MTOR, S6 kinase, STAT5, CAMKK, SYK (KSYK), LYN, P38A, CDK1, CK2A1, PKACA, IRAK4, and PKCB by analyzing data regarding phosphorylation in leukemic cells from said patient of one or more phosphorylation sites in one, two, three, four, five, six, seven, eight, nine, ten or more of iso2, Cot, PKCD, PKCA, PKCB, PKCG, PKCH, BRAF, MEK2, PDK1, CDK2, PTN6, D3 (PLD3), IQGAP1, GRB2, RHOA, RHOG, and S10AB; and (2) by analyzing data regarding surface expression on leukemic cells obtained from said patient of a panel of cell surface marker proteins comprising one or more of CD3, CD7, CD11b, CD11c (integrin α-X, ITAX), CD14, CD15, CD16, CD18 (integrin β, ITB2), CD19, CD33, CD34, CD35 (CR1), CD38, CD44, CD45, CD64, CD97, CD117, CD123, CD180, CD184, HLA-C (1C02), APOBR, platelet membrane receptor Gi24 (VSIR), and HLA-DR, and / or cell surface proteins expressed in conjunction with said one or more cell surface marker proteins; wherein phosphorylation of said one or more phosphorylation sites or a higher level of phosphorylation compared to undifferentiated myeloblasts or healthy cell types, and expression of said panel of cell surface marker proteins and / or cell surface proteins expressed in conjunction with said cell surface marker proteins on said leukemia cells at a higher level compared to undifferentiated myeloblasts or healthy cell types, indicates an advanced differentiation state; and (b) predicting that the patient's acute myeloid leukemia may be effectively treated with the kinase pathway inhibitor if the differentiation state of the leukemia cells is advanced; A method comprising:

2. 1. A method for screening a plurality of patients having acute myeloid leukemia to determine whether acute myeloid leukemia in one or more of the plurality of patients can be effectively treated with a kinase pathway inhibitor, which inhibits a signaling pathway involved in cell proliferation or cell survival, the method comprising the steps of: (a) determining for each patient the differentiation state of said patient's leukemic cells by: (1) FES, PKS, and protein kinase C isoforms, PKA, PAK, STK10, GSK3A, RSK2, RAS, RAF, MEK, ERK, PI3K, AKT, MTOR, S6 kinase, STAT5, CAMKK, SYK (KSYK), LYN, P38A, CDK1, CK2A1, PKACA, IRAK4, PKCB analyzing data regarding phosphorylation in leukemic cells from said patient of one or more phosphorylation sites in one, two, three, four, five, six, seven, eight, nine, ten or more of iso2, Cot, PKCD, PKCA, PKCB, PKCG, PKCH, BRAF, MEK2, PDK1, CDK2, PTN6, D3 (PLD3), IQGAP1, GRB2, RHOA, RHOG, and S10AB; and (2) analyzing data regarding surface expression on leukemic cells obtained from said patient of a panel of cell surface marker proteins comprising one or more of CD3, CD7, CD11b, CD11c (integrin alpha-X, ITAX), CD14, CD15, CD16, CD18 (integrin beta, ITB2), CD19, CD33, CD34, CD35 (CR1), CD38, CD44, CD45, CD64, CD97, CD117, CD123, CD180, CD184, HLA-C (1C02), APOBR, platelet membrane receptor Gi24 (VSIR), and HLA-DR, and / or a panel of cell surface proteins expressed in conjunction with said one or more cell surface marker proteins; Phosphorylation of said one or more phosphorylation sites or at a higher level compared to undifferentiated myeloblasts or healthy cell types, and expression of said panel of cell surface marker proteins and / or a panel of cell surface proteins expressed in conjunction with said cell surface marker proteins at a higher level on said leukemic cells compared to undifferentiated myeloblasts or healthy cell types, indicates an advanced differentiation state; and (b) identifying one or more patients having leukemic cells in an increasingly differentiated state as having leukemic cells suitable for effective treatment with said kinase pathway inhibitor. A method comprising:

3. 3. The method of claim 1 or 2, wherein the panel of cell surface marker proteins comprises: (i) any one, two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen, or fifteen, or sixteen, or seventeen, or eighteen, or all, of CD11b, CD11c (integrin α-X, ITAX), CD14, CD15, CD16, CD18 (integrin β, ITB2), CD33, CD35 (CR1), CD38, CD44, CD45, CD64, CD97, CD123, CD180, HLA-C (1C02), APOBR, platelet membrane receptor Gi24 (VSIR), and HLA-DR; or (ii) any one, two, three, four, five, six, seven, eight, nine, ten, or all of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; or (iii) any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all of CD3, CD7, CD11b, CD14, CD15, CD16, CD19, CD33, CD34, CD38, CD44, CD45, CD64, CD117, CD123, CD184, and HLA-DR.

4. 4. The method of any one of claims 1 to 3, wherein the panel of cell surface marker proteins comprises CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR.

5. The method according to any one of claims 1 to 4, wherein the phosphorylation site is a phosphorylation site in: (i) any one, two, three, or four of PKC, ERK, PAK1, and p38α; or (ii) any one, two, three, four, or five of PKCD, PKCA, PKACA, IRAK4, and CK2A1; or (iii) any one, two, three, four, five, or six of MAPK1, MAPK2, AKT, AKT1S1, MAP2K1, and MAP2K2; or (iv) any one, two, three, four, five, six, seven, eight, or nine of GSK3A, PRKCA, PRKCB, PRKCD, STK10, PAK1, PAK2, MAPK1, and MAPK3.

6. The method of any one of claims 1 to 5, wherein the phosphorylation site is: (i) any one, two, three, four, five, six, seven, eight, nine, ten, or more than ten phosphorylation sites listed in Table 1; or (ii) any one, two, three, four, five, six, seven, eight, nine, ten, or more than ten phosphorylation sites listed in Table 2; or (iii) PAK1 at S144, PAK2 at S141, MAPK1 at Y187, MAPK1 at T185, RPS6KA1 at S380, MAPK3 at T202, MAPK3 at Y204, MAP3K3 at S166, SYK at S295 and S297, IRAK3 at S110, PKN1 (379-396+phosphoST), STK10 (447-464+phosphoST), S410 RIPK3 at T218, PRKCD at T295, PRKCD at Y313, PRKCD at T507, PRKCD at T645, PRKCD at S664, PRKCD at T2638, MARK2 at S535, MAP3K2 at S535, PRKD2 (710-730+phosphoY), NRK at s805, PRKAR2A at S58, ZAK (591-616+phosphoY) ST), MAP4K4 at S900, CDK9 at S347, RPS6KA4 (681-699 + 2 phosphoST), MAST3 (1254-1274 + phosphoST), NEK9 (10-39 + phosphoST), GSK3A (19-50 + phosphoST), RPS6KA3 at S369, RIPK2 at S531, AAK1 at T606, TYK2 at Y292, PDPK2 at S214, PRKA or any one, two, three, four, five, six, seven, eight, nine, ten, or more than ten of: A1 (3-8+phosphoST), STK11P at S772, BAZ1B at S1468, CLK1 at S140, MAP4K2 at S328, WNK1 (1996-2021+phosphoST), CDK11A at S271, FES at Y713, and / or TNIK at S769; or (iv) PAK1 at S144, PAK2 at S141, MAPK1 at Y187 and / or T185, and RPS6KA1 at S380; or (v) MAPK1 at Y187, PAK2 at S141, and PRKCD at Y313; or (vi) FES at Y713, MAPK3 at T202 / Y204, MAPK1 at T185 / Y187, PAK1 at S144, MEK1 at S222, PAK2 at S141, and PRKCD at S645; or (vii) GSK3A at S21, and / or PRKCD at T507, T295, T218, Y313, T507 and / or S664, and / or STK10 at S20 and / or S13, and / or PAK1 at S144, and / or PAK2 at S141; or (viii) one or more phosphorylation sites in MAPK1 at Y187 and / or T185, and in MAPK3 at T202 and / or Y204, and in GSK3A at S21.

7. The method of any one of claims 1 to 6, wherein step (a) comprises: (i) determining the differentiation state of the patient's leukemic cells by analyzing data regarding phosphorylation of one or more phosphorylation sites in MAPK1 and / or MAPK3 in the leukemic cells obtained from the patient; and (ii) determining the differentiation state of the patient's leukemic cells by analyzing data relating to the surface expression on leukemic cells obtained from the patient of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; Here, phosphorylation or high levels of phosphorylation at said one or more phosphorylation sites compared to undifferentiated myeloblasts or healthy cell types, and / or high levels of expression of said group of CD markers on said leukemia cells compared to undifferentiated myeloblasts or healthy cell types, indicates an advanced differentiation state.

8. The method of any one of claims 1 to 7, wherein the kinase pathway inhibitor is a kinase pathway inhibitor selected from a MEK inhibitor, a FLT3 / PKC inhibitor, and a PAK inhibitor.

9. the kinase pathway inhibitor is a MEK inhibitor; Step (a) further comprises determining the mutation status of NRAS, KRAS, HRAS, or BRAF in the leukemic cells obtained from the patient by analyzing data relating to the genotype of the leukemic cells; and 9. The method of any one of claims 1 to 8, wherein step (b) comprises predicting that the patient's acute myeloid leukemia may be effectively treated with the kinase pathway inhibitor if the leukemia cells have an advanced differentiation state or if the leukemia cells have an activating mutation in one of NRAS, KRAS, HRAS, or BRAF.

10. Step (a) is (i) determining the mutation status of FLT3 in leukemic cells obtained from said patient by analyzing data relating to the genotype of said leukemic cells; and / or (ii) determining activation in said leukemia cells of FLT-3-driven signaling pathways involved in cell proliferation or cell survival other than the RAS-RAF-MEK-ERK pathway by analyzing data on activation markers of FLT3-driven kinase signaling pathways in said leukemia cells; and / or (iii) determining the level of phosphorylation of one or both of TOP2A and / or KDM5C in said leukemic cells by analyzing data regarding phosphorylation of TOP2A and / or KDM5C in said leukemic cells; Further comprising: In step (b), further comprising: if FLT3 does not have an activating mutation in said leukemia cells, or said FLT3-driven kinase signaling pathway is not activated in said leukemia cells, or TOP2A and / or KDM5C are not phosphorylated in said leukemia cells or are phosphorylated at a lower level compared to undifferentiated myeloblasts or healthy cell types, the kinase pathway inhibitor predicted to be capable of effectively treating acute myeloid leukemia in said patient is a MEK inhibitor; The method according to claim 8.

11. Step (a) is (i) determining the differentiation state of the patient's leukemic cells by analyzing data regarding the genotype of the leukemic cells, and / or determining the NRAS, KRAS, HRAS, or BRAF mutation status in the leukemic cells obtained from the patient; and (ii) determining the mutation status of FLT3 in leukemic cells obtained from said patient by analyzing data relating to the genotype of said leukemic cells; and / or determining activation in said leukemia cells of FLT-3-driven kinase signaling pathways involved in cell proliferation or cell survival other than the RAS-RAF-MEK-ERK pathway by analyzing data relating to activation markers of FLT-3-driven kinase signaling pathways in said leukemia cells; and / or determining the level of phosphorylation of one or both of TOP2A and / or KDM5C in said leukemic cells by analyzing data regarding phosphorylation of TOP2A and / or KDM5C in said leukemic cells; Further comprising: In step (b), further comprising: (1) the differentiation state of the leukemia cells is advanced or the leukemia cells have an activating mutation in one of NRAS, KRAS, HRAS, or BRAF; and (2) if FLT3 does not have an activating mutation in the leukemia cells, or the FLT3-driven kinase signaling pathway is not activated, or TOP2A and / or KDM5C are not phosphorylated or are phosphorylated at a lower level in the leukemia cells compared to undifferentiated myeloblasts or healthy cell types, the kinase pathway inhibitor predicted to be capable of effectively treating acute myeloid leukemia in said patient is a MEK inhibitor; 10. The method of claim 9.

12. The method of claim 10 or 11, wherein the activity marker of the FLT3-driven kinase signaling pathway comprises one or more phosphorylation sites that are selectively phosphorylated by the FLT3-driven kinase signaling pathway, and the data regarding the activity marker comprises data regarding the phosphorylation of the one or more phosphorylation sites included in the activity marker, and phosphorylation of the one or more phosphorylation sites included in the activity marker or a higher level of phosphorylation compared to undifferentiated myeloblasts or healthy cell types indicates that the FLT3-driven kinase signaling pathway is activated.

13. The method of claim 12, wherein the one or more phosphorylation sites contained in the activity marker include phosphorylation sites in one or both of STAT5A and CAMKK1.

14. The method of any one of claims 10 to 13, wherein the data regarding phosphorylation of TOP2A and / or KDM5C in the leukemic cells comprises data regarding phosphorylation of TOP2A and KDM5C.

15. The method of any one of claims 1 to 8, wherein the kinase pathway inhibitor is a FLT3 / PKC pathway inhibitor, and step (a) comprises determining the differentiation state of the patient's leukemic cells by: (i) analyzing data regarding phosphorylation of phosphorylation sites in one or more of GSK3A, PRKCA, PRKCB, PRKCD, STK10, PAK1, PAK2, MAPK1, and / or MAPK3 in leukemic cells obtained from said patient; and (ii) analyzing data regarding the surface expression on leukemic cells obtained from said patient of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD4PKC5, CD64, CD123, and HLA-DR; wherein either phosphorylation in the leukemic cells of one or more of GSK3A, PRKCA, PRKCB, PRKCD, STK10, PAK1, PAK2, MAPK1, and / or MAPK3 or high levels of phosphorylation compared to undifferentiated myeloblasts or healthy cell types; or expression by the leukemic cells of the group of CD markers at high levels compared to undifferentiated myeloblasts or healthy cell types, indicates an advanced differentiation state.

16. 1. A method for predicting the efficacy of midostaurin in treating acute myeloid leukemia in an individual patient, comprising the steps of: (a)(i) analyzing data regarding expression of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR on the surface of the patient's leukemic cells to determine whether the group of CD markers is expressed at a higher level on the surface of the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types; and (ii) analyzing data regarding phosphorylation in said leukemic cells at one or more phosphorylation sites selected from GSK3A at pS21; PRKCD at Y313, pT507, pT295, pT218, and / or pS664 of PRKCD; STK10 at pS20 and / or pS13 of STK10; PAK1 at pS144 of PAK1; PAK2 at pS141 of PAK2; MAPK1 at Y187 and / or T185; and MAPK3 at T202 and / or Y204 to determine whether one or more of said phosphorylation sites are phosphorylated or are phosphorylated at an increased level in the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types; and (b) predicting that the patient's acute myeloid leukemia may be effectively treated with midostaurin if the group of CD markers is expressed at a higher level on the surface of the patient's leukemia cells compared to undifferentiated myeloblasts or healthy cell types, and if one or more of the phosphorylation sites in the leukemia cells are phosphorylated or are phosphorylated at a higher level compared to undifferentiated myeloblasts or healthy cell types.

17. 1. A method for predicting the efficacy of trametinib in treating acute myeloid leukemia in an individual patient, comprising the steps of: (a)(i) analyzing data regarding expression of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR on the surface of the patient's leukemic cells to determine whether the group of CD markers is expressed at a higher level on the surface of the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types; and (ii) analyzing data regarding phosphorylation in the leukemic cells at one or more MEK-associated phosphorylation sites selected from MAPK1 at Y187 and / or T185, and MAPK3 at T202 and / or Y204 to determine whether one or more of the MEK-associated phosphorylation sites are phosphorylated or phosphorylated at a higher level in the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types; and (iii) analyzing the data regarding the presence or absence of any activating mutations in any one or more of NRAS, KRAS, HRAS, or BRAF in said leukemic cells of said patient to determine whether an activating mutation is present in any one or more of NRAS, KRAS, HRAS, or BRAF in said leukemic cells of said patient; and (b)(i) analyzing data regarding phosphorylation in the leukemic cells at one or more marker sites selected from STAT5A at pS780 and / or pS128, CAMKK1 at pS548, TOP2A at pS1213, and KDM5C at pS317 to determine whether the marker sites are phosphorylated or phosphorylated at an elevated level in the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types; and (ii) analyzing the data regarding the presence or absence of any activating mutations in FLT3 in said leukemia cells of said patient to determine whether an activating mutation is present in FLT3 in said leukemia cells of said patient; and (c) (A) the group of CD markers are expressed at a higher level on the surface of the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types, one or more of the MEK-associated phosphorylation sites in the leukemic cells are phosphorylated or are phosphorylated at a higher level compared to undifferentiated myeloblasts or healthy cell types, and / or there is an activating mutation in any one or more of NRAS, KRAS, HRAS, or BRAF in the leukemic cells of the patient; and (B) if the marker site is not phosphorylated or is not phosphorylated at a high level in the patient's leukemia cells compared to undifferentiated myeloblasts or healthy cell types, and there is no activating mutation in FLT3 in the patient's leukemia cells, Predicting that the patient's acute myeloid leukemia can be effectively treated with trametinib.

18. 1. A method for screening a plurality of patients having acute myeloid leukemia to determine whether the acute myeloid leukemia of any one or more of said plurality of patients can be effectively treated with midostaurin, the method comprising the steps of: (a) for each patient, (i) analyzing data regarding expression of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR on the surface of the patient's leukemic cells to determine whether the group of CD markers is expressed at a higher level on the surface of the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types; and (ii) analyzing data regarding phosphorylation in said leukemic cells at one or more phosphorylation sites selected from GSK3A at pS21; PRKCD at Y313, pT507, pT295, pT218, and / or pS664 of PRKCD; STK10 at pS20 and / or pS13 of STK10; PAK1 at pS144 of PAK1; PAK2 at pS141 of PAK2; MAPK1 at Y187 and / or T185; and MAPK3 at T202 and / or Y204 to determine whether one or more of said phosphorylation sites are phosphorylated or are phosphorylated at an increased level in the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types; and (b) identifying any one or more patients having leukemic cells that express said group of CD markers and that exhibit phosphorylation at one or more of said phosphorylation sites in said leukemic cells or high levels of phosphorylation compared to undifferentiated myeloblasts or healthy cell types as having a leukemia suitable for effective treatment with midostaurin.

19. 1. A method of screening a plurality of patients having acute myeloid leukemia to determine whether the acute myeloid leukemia of any one or more of said plurality of patients can be effectively treated with trametinib, comprising the steps of: (a) for each patient, (i) analyzing data regarding expression of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR on the surface of the patient's leukemic cells to determine whether the group of CD markers is expressed at a higher level on the surface of the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types; and (ii) analyzing data regarding phosphorylation in the leukemic cells at one or more MEK-associated phosphorylation sites selected from MAPK1 at Y187 and / or T185, and MAPK3 at T202 and / or Y204 to determine whether one or more of the MEK-associated phosphorylation sites are phosphorylated or phosphorylated at a higher level in the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types; and (iii) analyzing the data regarding the presence or absence of any activating mutations in any one or more of NRAS, KRAS, HRAS, or BRAF in said leukemic cells of said patient to determine whether an activating mutation is present in any one or more of NRAS, KRAS, HRAS, or BRAF in said leukemic cells of said patient; and (b) for each patient, (i) analyzing data regarding phosphorylation in the leukemic cells at one or more marker sites selected from STAT5A at pS780 and / or pS128, CAMKK1 at pS548, TOP2A at pS1213, and KDM5C at pS317 to determine whether the marker sites are phosphorylated or are phosphorylated at a higher level in the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types; and (ii) analyzing the data regarding the presence or absence of any activating mutations in FLT3 in said leukemia cells of said patient to determine whether an activating mutation is present in FLT3 in said leukemia cells of said patient; and (c) identifying any one or more patients as having a leukemia suitable for effective treatment with trametinib if: (A) the group of CD markers are expressed at a higher level on the surface of the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types, one or more of the MEK-associated phosphorylation sites in the leukemic cells are phosphorylated or are phosphorylated at a higher level compared to undifferentiated myeloblasts or healthy cell types, and an activating mutation is present in any one or more of NRAS, KRAS, HRAS, or BRAF in the leukemic cells of the patient; and (B) the marker site is not phosphorylated or is not phosphorylated at high levels in the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types, and there are no activating mutations in FLT3 in the patient's leukemic cells.

20. 1. A kinase pathway inhibitor for use in a method of treating acute myeloid leukemia in an individual patient, the kinase pathway inhibitor inhibiting a signal transduction pathway involved in cell proliferation or cell survival, the treatment comprising: (a) determining the differentiation state of leukemic cells of said patient by: (1) FES, PKS, and protein kinase C isoforms, PKA, PAK, STK10, GSK3A, RSK2, RAS, RAF, MEK, ERK, PI3K, AKT, MTOR, S6 kinase, STAT5, CAMKK, SYK (KSYK), LYN, P38A, CDK1, CK2A1, PKACA, IRAK4, PKCB analyzing data regarding phosphorylation in leukemic cells from said patient of one or more phosphorylation sites in any one, two, three, four, five, six, seven, eight, nine, ten or more of iso2, Cot, PKCD, PKCA, PKCB, PKCG, PKCH, BRAF, MEK2, PDK1, CDK2, PTN6, D3 (PLD3), IQGAP1, GRB2, RHOA, RHOG, and S10AB; and (2) analyzing data regarding surface expression on leukemic cells obtained from said patient of a panel of cell surface marker proteins comprising one or more of CD3, CD7, CD11b, CD11c (integrin alpha-X, ITAX), CD14, CD15, CD16, CD18 (integrin beta, ITB2), CD19, CD33, CD34, CD35 (CR1), CD38, CD44, CD45, CD64, CD97, CD117, CD123, CD180, CD184, HLA-C (1C02), APOBR, platelet membrane receptor Gi24 (VSIR), and HLA-DR, and / or a panel of cell surface proteins expressed in conjunction with said one or more cell surface marker proteins; wherein phosphorylation of said one or more phosphorylation sites or a higher level of phosphorylation compared to undifferentiated myeloblasts or other healthy cell types, and expression of said cell surface marker protein, and / or any panel of cell surface proteins expressed in conjunction with the cell surface marker protein, on said leukemic cells compared to undifferentiated myeloblasts or healthy cell types, indicates an advanced differentiation state; and (b) administering said kinase pathway inhibitor to said patient for the treatment of acute myeloid leukemia if the differentiation state of said leukemia cells is advanced. A kinase pathway inhibitor comprising:

21. 1. A kinase pathway inhibitor for use in a method of treating acute myeloid leukemia in a patient, the kinase pathway inhibitor inhibiting a signaling pathway involved in cell proliferation or cell survival, the patient comprising: (a) FES, PKS, and protein kinase C isoforms, PKA, PAK, STK10, GSK3A, RSK2, RAS, RAF, MEK, ERK, PI3K, AKT, MTOR, S6 kinase, STAT5, CAMKK, SYK (KSYK), LYN, P38A, CDK1, CK2A1, PKACA, IRAK4, and PKCB. phosphorylation or high levels of phosphorylation at one or more phosphorylation sites in any one, two, three, four, five, six, seven, eight, nine, ten, or more of iso2, Cot, PKCD, PKCA, PKCB, PKCG, PKCH, BRAF, MEK2, PDK1, CDK2, PTN6, D3 (PLD3), IQGAP1, GRB2, RHOA, RHOG, and S10AB compared to undifferentiated myeloblasts or healthy cell types; and having leukemic cells in an increasingly differentiated state characterized by high levels of surface expression of cell surface marker proteins, including one or more of CD3, CD7, CD11b, CD11c (integrin alpha-X, ITAX), CD14, CD15, CD16, CD18 (integrin beta, ITB2), CD19, CD33, CD34, CD35 (CR1), CD38, CD44, CD45, CD64, CD97, CD117, CD123, CD180, CD184, HLA-C (1C02), APOBR, platelet membrane receptor Gi24 (VSIR), and HLA-DR, and / or a panel of cell surface proteins expressed in conjunction with said one or more cell surface marker proteins, compared to undifferentiated myeloblasts or healthy cell types; Kinase pathway inhibitors.

22. 1. Midostaurin for use in a method of treating acute leukemia in a patient, said patient having leukemia cells having an advanced differentiation state defined by: (i) high levels of surface expression on the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; and (ii) the following: GSK3A pS21; pY313, pT507, pT295, pT218, and / or pS664 of PRKCD; pS20 and / or pS13 of STK10; PAK1 pS144; PAK2 pS141; Y187 and / or T185 of MAPK1; and T202 and / or Y204 of MAPK3 in said leukemic cells of said patient or at high levels compared to undifferentiated myeloblasts or healthy cell types.

23. 2. Trametinib for use in a method of treating acute leukemia in a patient, wherein the patient has leukemic cells with an advanced differentiation state defined by: (a)(i) high level surface expression on the patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types of a group of CD markers consisting of CD11b, CD14, CD15, CD16, CD33, CD38, CD44, CD45, CD64, CD123, and HLA-DR; (ii) a high level of phosphorylation in said patient's leukemic cells compared to undifferentiated myeloblasts or healthy cell types of any one or more of Y187 and / or T185 of MAPK1; and T202 and / or Y204 of MAPK; and (iii) an activating mutation in any one or more of NRAS, KRAS, HRAS, or BRAF in the leukemia cells of the patient; and (b) (i) phosphorylation or high levels of phosphorylation at one or more of pS780 and pS128 of STAT5A, pS548 of CAMKK1, pS1213 of TOP2A, and pS317 of KDM5C compared to undifferentiated myeloblasts or healthy cell types; and (ii) the absence of any activating mutations in FLT3 in said leukemic cells of said patient.

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