WO2014087018A1

WO2014087018A1 - Markers for responsiveness to an inhibitor of flt3

Info

Publication number: WO2014087018A1
Application number: PCT/EP2013/075970
Authority: WO
Inventors: Christoph Schaab; Klaus Godl; Felix S. OPPERMANN; Martin KLAMMER; Andreas Tebbe; Hubert Serve
Original assignee: Evotec (München) Gmbh
Priority date: 2012-12-07
Filing date: 2013-12-09
Publication date: 2014-06-12

Abstract

The present invention relates to methods of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, like quizartinib (AC220). The methods comprise determining the phosphorylation status of B-cell lymphoma/leukemia 11A protein (BCL1 1A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in a sample of a patient, wherein the phosphorylation status is indicative of responsiveness to FLT3 inhibitor. The patient can suffer from neoplasia, like AML. Also the therapeutic use of FLT3 inhibitors (like quizartinib (AC220)) in the treatment of patients determined to have a specific phosphorylation status is provided.

Description

Markers for responsiveness to an inhibitor of FLT3

The present invention relates to methods of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, like quizartinib (AC220). The methods comprise determining the phosphorylation status of B-cell lymphoma/leukemia 11A protein (BCLl lA), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in a sample of a patient, wherein the phosphorylation status is indicative of responsiveness to FLT3 inhibitor. The patient can suffer from neoplasia, like AML. Also the therapeutic use of FLT3 inhibitors (like quizartinib (AC220)) in the treatment of patients determined to have a specific phosphorylation status is provided.

Acute Myeloid Leukemia (AML) results from a combination of oncogenic events that can involve multiple signal transduction pathways including mutation-induced activation of tyrosine kinases. Kinase inhibitors are increasingly studied as promising targeted approaches either alone or in combination with other agents. However, only subsets of patients respond to respective targeted therapies. Response prediction tests, which are currently used in the clinic, are often based on markers that are linked to the drug's target. The maybe best-known example is the test on HER2/neu overexpression using immunohistochemistry or fluorescent in situ hybridization for predicting response to treatment with trastuzumab (Herceptin®, Roche) (see Cobleigh, Vogel et al. 1999; Ross and Fletcher 1999). However, the expression or mutational status of singleton markers might not always be sufficient to predict a response. Recently, it has been shown for example that the use of genotyping alone may be limited in predicting response of melanoma to targeted therapies (Passeron, Lacour et al. 2011). In two recent studies phosphoproteomics data has been used to identify predictive markers for phosphatidylinositol 3-kinase (PI3K) inhibitors (Andersen, Sathyanarayanan et al. 2010) and the multi-kinase inhibitor dasatinib (Klammer, Kaminski et al. 2012). In contrast to the data provided in accordance with the present invention, both studies analysed cell lines. By directly analysing clinical samples, the present invention advantageously omits the translation from the pre-clinic to the clinic. In recent years, advances in sample processing, mass spectrometry, and computer algorithms for the analyses of proteomics data have enabled the application of mass spectrometry-based proteomics to monitor phosphorylation events in a global and unbiased manner (Olsen, Blagoev et al. 2006; Macek, Mann et al. 2009; Schaab 2011). These methods have become sufficiently sensitive and robust to identify and quantify thousands of phosphorylation sites in a single experiment. Furthermore, spiking-in a SILAC- (stable-isotope labeling by amino acids in cell culture) reference sample (Super-SILAC) allows for the precise quantification of phosphorylation events also in in- vivo samples (Geiger, Cox et al. 2010).

Internal tandem duplication (ITD) of FLT3 is one of the most common mutations in AML. It causes constitutive activation of FLT3. AC220 (Quizartinib®, Ambit) is a selective inhibitor of the receptor-type tyrosine-protein kinase FLT3 (Zarrinkar, Gunawardane et al. 2009) that is currently under development for the treatment of AML. In a recent phase II open-label study, patients with relapsed AML were treated with AC220. A planned interim analysis (Cortes, Perl et al. 2011) showed that the complete remission rate (including CRp and CRi) in FLT3-1TD positive patients was 43% (23/53). The corresponding partial remission rate was 28% (15/53). In a previous phase I study, the total response rate (CR+PR) in FLT3-ITD negative patients was 20% (9/45) (Cortes, Foran et al. 2009). Thus, although the FLT3-ITD mutation status correlates with response, it does not perfectly stratify the patients into responder and non-responder. Still 28% of the FLT3-ITD positive patients fail to respond. Even more serious is the fact that by selecting only FLT3-ITD positive patients for treatment with AC220, roughly 20% of the FLT3-ITD negative patients, who do respond, would be missed.

Herein a phosphorylation signature is provided that predicts clinical response with higher accuracy than the FLT3-ITD status.

Thus, the technical problem underlying the present invention is the provision of reliable means and methods to determine responsiveness of proliferative diseased cells and/or patients to an inhibitor of an FMS-like tyrosine kinase 3 (FLT3).

The technical problem is solved by provision of the embodiments characterized in the claims. Accordingly, the present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of one or more of B-cell lymphoma/leukemia 11A protein (BCLl lA), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention also provides for therapy of patients with FMS-Hke tyrosine kinase 3 (FLT3) inhibitor, wherein a proliferative diseased cell of a sample of the patient is determined to have one or more of B-cell lymphoma/leukemia 11A protein (BCLl lA), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), and/or Ran- binding protein 3 (RANBP3) not phosphorylated; and/or to have GTPase regulator (RP3) phosphorylated. The terms "phosphorylated" and "not phoshorylated" as used herein refer to the presence and absence, respectively, of phosphorylation at one or more phosphorylation sites of one or more of B-cell lymphoma/leukemia 11A protein (BCLl lA), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3). The one or more phosphorylation sites are one or more of the amino acid residues, typically one or more of serine and/or threonine residues, of one or more of B-cell lymphoma/leukemia 1 1A protein (BCLl lA), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3).

For example, one or more of B-cell lymphoma/leukemia 11A protein (BCLl lA), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), and/or Ran-binding protein 3 (RANBP3) are not phosphorylated at one or more phosphorylation sites as defined herein; and/or GTPase regulator (RP3) is phosphorylated at one or morephosphorylation sites as defined herein. Phosphorylation sites are, for example, one or more of S630 of BCLl lA, S205 of BCLl lA, S328 of BCLl lA, S608 of BCLl lA, S625 of BCLl lA, S718 of BCLl lA, T208 of BCLl lA, S160 of EEPDl, S173 of EEPDl, S25 of EEPDl, S554 of EEPDl, S458 of LMNl , S22 of LMNl, S277 of LMNl , S398 of LMNl, T10 of LMNl, S333 of RANBP3, S108 of RANBP3, S961 of RP3 and/or S518 of RP3. Phosphorylation sites refer in particular to one or more of S630 of BCLl lA, S160 of EEPDl , S458 of LMNl, S333 of RANBP3, and/or S961 of RP3 and/or to one or more of the following correlated phosphorylation sites S205 of BCLl lA, S328 of BCLl lA, S608 of BCLl lA, S625 of BCLl lA, S718 of BCLl lA, S25 of EEPDl, and/or S554 of EEPDl.

The present invention provides a method of treating a patient, said method comprising selecting a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is determined

(i) to have one or more of B-cell lymphoma/leukemia 11A protein (BCLl lA), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), and/or Ran-binding protein 3 (RANBP3) not phosphorylated; and/or

(ii) to have GTPase regulator (RP3) phosphorylated; and

administering to the patient an effective amount of an FMS-like tyrosine kinase 3 (FLT3) inhibitor.

In other words, a neoplasia patient is selected for an FMS-like tyrosine kinase 3 (FLT3) inhibitor therapy, if a proliferative diseased cell of a sample of the patient is determined

(ii) to have GTPase regulator (RP3) phosphorylated.

The present invention provides an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient

(i) is not phosphorylated at one or more phosphorylation site of one or more of B-cell lymphoma/leukemia 11A protein (BCLUA), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), and/or Ran- binding protein 3 (RANBP3); and/or

(ii) is phosphorylated at one or more phosphorylation site of GTPase regulator (RP3).

The "one or more phosphorylation sites" refer in particular to one or more of S630 of BCL1 1A, S160 of EEPDl, S458 of LMNl, S333 of RANBP3, and/or S961 of RP3 and/or to one or more of the following correlated phosphorylation sites S205 of BCLl lA, S328 of BCLl lA, S608 of BCLl lA, S625 ofBCLUA, S718 ofBCLl 1A, S25 of EEPDl, and/or S554 of EEPDl . The present invention provides an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient

(i) is not phosphorylated at one or more phosphorylation site of one or more of B-cell lymphoma/leukemia 11A protein (BCLl lA), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), and/or Ran- binding protein 3 (RANBP3); and/or

(ii) is phosphorylated at one or more phosphorylation site of GTPase regulator (RP3),

wherein the treatment comprises determining the phosphorylation status at one or more phosphorylation sites of one or more of B-cell lymphoma/leukemia 1 1A protein (BCLl lA), Lamin A/C (LMN1), endonuclease/exoiruclease/phosphatase family domain-containing protein 1 (EEPD1), Ra -binding protein 3 (RANBP3) and/or GTPase regulator (RP3) in a sample of a patient.

Determining the phosphorylation status refers to determining the absence or presence of phosphorylation at one or more phosphorylation sites (and likewise decrease or increase of phosphorylation at one or more phosphorylation sites).

It is to be understood that a neoplasia patient is to be treated with an FMS-like tyrosine kinase 3 (FLT3) inhibitor, if a proliferative diseased cell of a sample of the patient is determined

(i) to have one or more of B-cell lymphoma/leukemia 1 1A protein (BCLl lA), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), and/or Ran-binding protein 3 (RANBP3) not phosphorylated; and/or

(ii) to have GTPase regulator (RP3) phosphorylated.

The below explanations and definitions in relation to "proliferative diseased cell(s)", "FMS-like tyrosine kinase 3 (FLT3) inhibitor(s)", "phosphorylation status", "phosphorylation site" "endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1 )", "B-cell lymphoma leukemia 1 1A protein (BCLl lA)", "Ran-binding protein 3 (RANBP3)", "GTPase regulator (RP3)", "Lamin A C (LM 1)", "sample", "patient", and the like apply, mutatis mutandis, to the herein provided "method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor", "method of treating a patient" and "FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient". The term "determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor" as used herein refers in particular and primarily to "predicting whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor". Thus, these terms can be used interchangeably herein. The meaning of the term "determining responsiveness to therapy/drug" (like "determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor") is well known in the art and used accordingly herein. For example, it is well known in the art that predictive factors determine the responsiveness to a treatment (Lonning, Annals of Oncology 18, supp 8 (2007) (doi: 10.1093/aimonc/mdm260). Thus, predictive factors indicate which adjuvant therapy is most appropriate (Lonning, loc. cit.). It is therefore contemplated and preferred herein that the determination of the phosphorylation status and/or of the expression level of the herein provided markers is performed prior to (the start of) the treatment with the FMS-like tyrosine kinase 3 (FLT3) inhibitor. Accordingly, "predicting whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor" is preferably performed before the first cycle of therapy/treatment with the FMS-like tyrosine kinase 3 (FLT3) inhibitor. Thus, the herein provided markers are preferably used as predictive markers.

Yet, it is also envisaged that the herein provided markers can be used as monitoring markers. In other words, the markers can be used to monitor the response to an FMS-like tyrosine kinase 3 (FLT3) inhibitor after treatment has started (e.g. during treatment, encompassing treatment breaks). Thus, "determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor" as used herein can refer to "monitoring whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor".

It is to be understood that the proliferative diseased cell(s) to be evaluated/assessed/scrutinized may be part of a sample (like a bone marrow sample or a blood sample, like a peripheral blood sample). Also the phosphorylation status of cells other than "proliferative diseased cell(s)" from a given sample (like a a bone marrow sample or a blood sample, like a peripheral blood sample) may be evaluated/assessed/scrutinized without deferring from the gist of this invention. The sample comprising the at least one proliferative diseased cell can be obtained from a patient. Typically, patients suffering from a proliferative disease such as neoplasia (like leukemia, such as acute myoloid leukemia (AML)) have a pathologically (abnormally) increased cell proliferation of certain cell types, in particular of white blood cells (white blood cells are also termed "leukocytes"). The term "white blood cell(s)" is used interchangeably herein with the term "leukocyte(s)". Typically, neoplasia (like leukemia, such as acute myoloid leukemia (AML)) is characterized by/associated with a pathological (abnormal) increase of immature white blood cells (immature white blood cells are also termed "blast(s)"). The terms "immature white blood cell", "immature leukocyte" and "blast" are used interchangeably herein. Thus, the "proliferative diseased cell(s)" can, in accordance with the present invention, be (a) white blood cell(s)/(a) leukocyte, such as (an) "immature white blood cell(s)"/"immature leukocyte(s)"/"blast"(s).The term "a proliferative diseased cell" refers also to a plurality thereof, i.e. to "proliferative diseased cells". Accordingly, the terms "a proliferative diseased cell" and "proliferative diseased cells" can be used interchangeably herein.

As shown herein in Fig. 7, three markers (BCL11A, EEPD1 and LMN1) show a strong correlation in their phosphorylation in the respective bone marrow and peripheral blood samples. This suggests that the phosphorylation status as defined and explained herein can be measured and is predictive in both bone marrow and blood samples (like a peripheral blood sample).

Depending on the specific pathological condition, different specific "immature white blood cell(s)"/"immature leukocyte(s)"/"blast"(s) may be pathologically/abnormally increased.

For example, in leukemias, like myeloid leukemias, such as acute myoloid leukemia (AML), the myeloid stem cells usually become a type of immature white blood cell called myeloblasts (or myeloid blasts). The terms "myeloblast(s)" and "myeloid blast(s)" are used interchangeably herein. The myeloblasts in AML are abnormal and do not become healthy white blood cells. In relation to „AML" the "proliferative diseased cell(s)" can, in accordance with the present invention, be "myeloblast(s)"/"myeloid blast(s)".

For example, in leukemias, like lymphoid leukemias, such as acute lymphoid leukemia (ALL), too many stem cells become lymphoblasts, B lymphocytes, or T lymphocytes. These cells are also called "leukemia cells" in the art. These leukemia cells do not work like normal lymphocytes and are not able to fight infection very well. Also, as the number of leukemia cells increases in the blood and bone marrow, there is less room for healthy white blood cells, red blood cells, and platelets. In relation to„ALL" the "proliferative diseased cell(s)" can, in accordance with the present invention, be "lymphoblast(s)", "B lymphocyte(s)", and/or "T lymphocyte(s)". Likewise, in relation to„ALL" the "proliferative diseased cell(s)" can, in accordance with the present invention, be "leukemia cells".

The terms "acute myoloid leukemia" and "AML"; and "acute lymphoid leukemia" and "ALL", respectively, are used interchangeably herein.

As demonstrated in the appended example, the phosphorylation status (or "phosphosignature") of one or more of B-cell lymphoma 1 eukemia 11A protein (BCL11A), Lamin A C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), , Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in at least one proliferative diseased cell in a sample from a patient indicates whether the diseased cell and, correspondingly, the patient suffering from a disease that is characterized by such proliferative diseased cells, will respond to treatment with an F S-like tyrosine kinase 3 inhibitor. The phosphorylation status may be assessed in cellular lysates as well as in whole samples (like bone marrow samples, blood samples (like a peripheral blood sample) etc.).

The terms "FMS-like tyrosine kinase 3 (FLT3) inhibitor", "FLT3 inhibitor", "FMS-like tyrosine kinase 3 inhibitor", "inhibitor of FMS-like tyrosine kinase 3 (FLT3)", "inhibitor of FLT3", "inhibitor of FMS-like tyrosine kinase 3" are used interchangeably herein. The terms "endonuclease/exonuclease/phosphatase family domain-containing protein 1" and "EEPD1"; "B- cell lymphoma/leukemia 11A protein" and "BCL1 1A"; "Ran-binding protein 3" and "RANBP3", "GTPase regulator" and "RP3"; and "Lamin A/C" and "LMN1", respectively, are used interchangeably herein.

The present invention solves the above identified technical problem since, as documented herein below and in the appended examples, it was surprisingly found that the phosphorylation status of the above protein/proteins can be used for a reliable determination of the responsiveness of a proliferative diseased cell or a patient to an FLT3 inhibitor (like AC220). The prediction accuracy was about 90 % (92 % in a first set and 88 % in a second validation set).

FLT3 inhibitors are used in the art to treat diseases that are characterized by activating FLT3 mutations. Activating FLT3 mutations induce a higher activity of the FLT3 protein. Thus, the use of FLT3 inhibitors is indicated to suppress increased activity of FLT3 in patients having such a FLT3 mutation. AC220 is a selective inhibitor of the receptor-type protein kinase FLT3. Accordingly, patients are determined in the art to be eligible for treatment with FLT3 inhibitors if activating FLT3 mutations, such as FLT3-ITD, are present. The drug responsiveness correlates with the presence of the FLT3-ITD mutation to a certain extent. However, roughly 28% of the FLT3-ITD positive patients do not respond (Cortes, Perl et al. 2011) and 20% of the FLT3-ITD negative patients do respond (Cortes, Foran et al. 2009). Thus, the presence of FLT3 mutations does not provide for a reliable determination of responding cells or patients. In contrast, virtually all patients determined to respond to FLT3 inhibitors in accordance with the present invention do indeed respond to FLT3 inhibitors.

This is surprising, because the herein provided phosphorylation markers do not play a role in the FLT3 signaling pathway. To the contrary, phosphorylation sites known to be involved in FLT3-ITD induced signalling cannot be used in the reliable determination of responders to an FLT3 inhibitor, as demonstrated in the appended example. Also phosphorylations of the protein kinases STAT5A and STAT5B at tyrosine sites Y694 (STAT5A) and Y699 (STAT5B), which are known to be activated by FLT3-ITD (Hayakawa, Towatari et al. 2000; Mizuki, Fenski et al. 2000; Choudhary, Olsen et al. 2009, Levis, Blood. 2002;99(11):3885~9L), show no differential phosphorylation between the responder and the non-responder classes (Fig. 2B).

It has been previously shown that inhibition of FLT3 leads to decreased phosphorylation of Bcl-2 antagonist of cell death (BAD) in cell lines with constitutively active FLT3 (Kim, Br J Haematol. 2006;134(5):500-9). Although this effect was confirmed in two primary AML samples with constitutively active FLT3, it has not be shown that the decreased phosphorylation of BAD correlates with inhibition of cell growth or induction of apoptosis. In fact, no significant difference between the phosphorylation levels of BAD in the group of responder compared to the group of non-responder was observed (see Example 5 and Fig. 9). Schaab (Blood 120: Abstract 786 (2012)) discloses phospoproteome analysis of AML bone marrow. Klammer (Mol Cell Proteom 11.9:651- 667 (2012)) discloses a phosphosignature that predicts dasatinib response in non-small cell lung cancer.

Levis et al. have demonstrated that the degree of cytotoxicity induced by an inhibitor of FLT3 (AG1295) correlates with the presence of a FLT3-ITD mutation in primary AML blasts (Levis Blood. 2001 ;98(3):885-7). However, as stated above, the presence of this mutation cannot reliably predict the response in patients to treatment with AC220 specifically, and with FLT3 inhibitors in general. Levis (loc. cit.) has further shown that inhibition of FLT3 results in decreased auto- phosphorylation of FLT3 in primary AML samples with constitutively active FLT3. However, Levis (loc. cit.) does not even speculate whether the degree of phosphorylation of FLT3 before or after treatment correlates with response.

Thus, the present invention provides for a reliable stratification of cells/patients responding to FLT3 inhibitors. This provides benefit for patients with FLT3 mutations, because patients with FLT3 mutations which are determined to be responsive according to the present invention can undergo FLT3 inhibitor therapy, while non-responders avoid side-effects of a likely ineffective therapy. Such non-responders may therefore choose treatment options other than FLT3 inhibitor therapy. The present invention also provides benefit for patients without FLT3 mutations (i.e. patients for which FLT3 inhibitor therapy is usually not contemplated in the art). Patients without FLT3 mutations which are determined to respond to FLT3 inhibitor therapy in accordance with the herein provided methods may undergo successful FLT3 inhibitor therapy.

Herein pre-treatment bone marrow aspirates from 21 patients enrolled in the AC220 trial were processed and analysed. Using a first collection of 12 samples, a phospho -signature of up to five phosphorylation sites was identified. The resulting prediction accuracy is 92%, the area under the receiver operating curve (AUROC) 89% as determined by cross-validation. In a second step, the signature was validated with additional nine samples that were not used during training. Seven out of nine patients were correctly classified. One of the misclassified patients (AML033) was classified as responder instead of as non-responder. Actually, the patient's FLT-ITD positive cells were sensitive. However the patient progressed with a FLT3 wild-type clone and wasn't called a responder. Depending on whether this ambiguous call is counted, the resulting accuracy is 78% (with AML033) or 88% (without AML033). In either case, the accuracy is high and in the range of the value determined by cross-validation.

Accordingly, the herein provided methods allow for the determination of the responsiveness to an FLT3 inhibitor with high accuracy. The accuracy of the herein provided methods is usually above 80 %. As shown herein in relation to AML patients, the accuracy is for example 70 %, 75 %, or more, or 80 %, 85 %, or 90 % or more. All five phosphorylation sites S630 of BCLl l A, S160 of EEPDl , S458 of LMNl , S333 of RANBP3 and S961 of RP3 were identified in previous phosphoproteomic studies. However no function has been described for them so far. None of the five marker phosphorylations or the corresponding proteins have been described as response prediction marker. For application of the biomarker signature in the clinic it is sufficient to detect and quantify at least one of the five herein provided phosphorylation sites, whereby the accuracy of the determination is usually the more reliable the more phosphorylation sites are assessed. Immunological methods or the mass- spectrometry-based multiple-reaction-monitoring (Kitteringham, Jenkins et al. 2009) can be applied in determining the phosphorylation status. These methods allow reproducible detection and quantification of given phosphorylations from relatively low sample amounts and can be routinely applied to large number of samples.

At least one of the marker phosphorylation, LMNl (S458), strongly correlates with the expression of the corresponding protein. The expression of LMNl can therefore be used as in the alternative to/independently of its phosphorylation status to determine whether a proliferative diseased cell or patient is responsive to an FLT3 inhibitor. The expression of LMNl can also be used in addition to/in combination with the phosphorylation status of LMNl to determine whether a proliferative diseased cell is responsive to an FLT3 inhibitor. The expression of LMNl can be used in addition to/in combination with the phosphorylation status of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), B-cell lymphoma/leukemia 1 1A protein (BCLl lA), Ran-binding protein 3 (RANBP3), GTPase regulator (RP3) and/or Lamin AJC (LMNl) to determine whether a proliferative diseased cell is responsive to an FLT3 inhibitor.

Herein provided is a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of one or more of B-cell lymphoma/leukemia 1 1A protein (BCLl lA), Lamin A/C (LMNl ), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor. The herein provided methods can comprise a step of obtaining a sample from a patient.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising the steps

(a) obtaining a sample to be evaluated;

(b) determining the phosphorylation status of one or more of B-cell lymphoma/1 eukemia 11 A protein (BCL11A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in a said sample,

wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The herein provided methods can comprise determining the phosphorylation status status of one or more of B-cell lymphoma/leukemia 1 1A protein (BCL1 1A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), , Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) . The phosphorylation status of only one, two, three, four or five of B-cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), and GTPase regulator (RP3) can be determined. The determination of the phosphorylation status of one or more of B-cell lymphoma/leukemia 1 1A protein (BCL11A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in any combination is envisaged herein. The phosphorylation status thereof may be determined simultaneously or subsequently (again in any combination) without deferring from the gist of the present invention.

For example, the methods of the present invention may comprise evaluating the phosphorylation status of

endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl) and B-cell lymphoma/leukemia 11 A protein (BCL11 A);

endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl) and Ran- binding protein 3 (RANBP3);

endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl) and GTPase regulator (RP3); endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1) and Lamin A/C (LMN1);

Ran-binding protein 3 (RANBP3) and GTPase regulator (RP3);

Ran-binding protein 3 (RANBP3) and Lamin A C (LMN1);

endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), B-cell lymphoma/leukemia 11A protein (BCLl 1 A) and Ran-binding protein 3 (RANBP3);

endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), B-cell lymphoma/leukemia 1 1 A protein (BCLl 1A) and GTPase regulator (RP3);

endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), B-cell lymphoma/leukemia 11A protein (BCLl 1A) and Lamin A/C (LMNl);

endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and GTPase regulator (RP3);

endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and Lamin A/C (LMNl);

endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), GTPase regulator (RP3) and Lamin A/C (LMNl);

B-cell lymphoma/leukemia 1 1 A protein (BCLl 1 A) and Ran-binding protein 3 (RANBP3);

B-cell lymphoma/leukemia 1 1 A protein (BCLl 1 A), and GTPase regulator (RP3);

B-cell lymphoma/leukemia 11 A protein (BCLl 1 A) and Lamin A C (LM l);

B-cell lymphoma/leukemia 11 A protein (BCLl 1 A), Ran-binding protein 3 (RANBP3), and GTPase regulator (RP3);

B-cell lymphoma/leukemia 11A protein (BCLl 1A), Ran-binding protein 3 (RANBP3), and Lamin A C (LMNl);

B-cell lymphoma/leukemia 11A protein (BCLl 1 A), GTPase regulator (RP3) and Lamin A C (LMNl);

Ran-binding protein 3 (RANBP3) and GTPase regulator (RP3);

Ran-binding protein 3 (RANBP3) and Lamin A/C (LMNl);

Ran-binding protein 3 (RANBP3), GTPase regulator (RP3) and Lamin A/C (LMNl); or

GTPase regulator (RP3) and Lamin A/C (LMNl);

and any combination thereof.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The method may further comprise determining the phosphorylation status of one or more of B-cell lymphoma/leukemia 11A protein (BCL11A), Ran-binding protein 3 (RANBP3), GTPase regulator (RP3) and/or Lamin A/C (LMN1) in a sample of a patient, wherein said status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

In a preferred embodiment, the present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of B-cell lymphoma/leukemia 11A protein (BCL1 1 A), in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The method may further comprise determining the phosphorylation status of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), GTPase regulator (RP3) and/or Lamin A/C (LM 1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Ran-binding protein 3 (RANBP3), in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The method may further further comprise determining the phosphorylation status of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), B-cell lymphoma/leukemia 11A protein (BCL11A), GTPase regulator (RP3) and/or Lamin A/C (LMN1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor. The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The method may further comprise determining the phosphorylation status of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), B-cell lymphoma/leukemia 1 1 A protein (BCL11 A), Ran-binding protein 3 (RANBP3), and/or Lamin A/C (LMN1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Lamin A/C (LMN1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The method may further comprise determining the phosphorylation status of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), B-cell lymphoma/leukemia 11A protein (BCLl lA), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

Exemplary combinations of two or more of endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPDl), B-cell lymphoma/leukemia 1 1A protein (BCLl lA), Ran-binding protein 3 (RANBP3), GTPase regulator (RP3) and Lamin A/C (LMN1), whose phosphorylation status is to be determined have been described above and can, mutatis mutandis, be used in the above described methods of the present invention.

It has been shown herein that the presence or absence of phosporylation at one or morephosphorylation site of one or more of B-cell lymphoma/leukemia 1 1A protein (BCLl lA), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protem 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) is indicative of responsiveness to a FLT3 inhibitor. The absence of phosporylation at one or morephosphorylation site of one or more of B-cell lymphoma/leukemia 1 1A protein (BCL1 1A), Lamin A C (LM 1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), and/or Ran- binding protein 3 (RANBP3), indicates responsiveness to the FMS-like tyrosine kinase 3 (FLT3) inhibitor. The presence of phosporylation at one or morephosphorylation site of GTPase regulator (RP3) indicates responsiveness to the FMS-like tyrosine kinase 3 (FLT3) inhibitor.

It is to be understood that a sample of a patient may comprise proliferative diseased cells (or cells other than proliferative diseased cells) that have a different phosphorylation status in one or more of B-cell lymphoma/leukemia 1 1A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3). A "different phosphorylation status" means in this context the absence or presence of phosphorylation (at one or more phosphorylation sites as described herein) of one or more of B-cell lymphoma/leukemia 11 A protein (BCL1 1 A), Lamin A C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) .

Even individual proteins in one proliferative diseased cell (or one cells other than a proliferative diseased cell) in a sample from a patient may have a different phosphorylation status in one or more of B-cell lymphoma/leukemia 11 A protein (BCLl lA), Lamin A C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3). For example, phosporylation may be present or absent at one or more phosphorylation sites in one or more of B-cell lymphoma/leukemia 11A protein (BCLl lA), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) and thus be indicative of responsiveness to a FLT3 inhibitor.

Without deferring from the gist of the present invention, a person skilled in the art will appreciate that the absence of phosporylation at one or more phosphorylation sites of one or more of B-cell lymphoma leukemia 11A protein (BCLl lA), Lamin A/C, endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), and Ran-binding protein 3 (RANBP3), and/or LMNl can indicate responsiveness to the FMS-like tyrosine kinase 3 (FLT3) inhibitor, if (preferably statistically significantly) more phosphorylation sites of one or more of these proteins are not phosphorylated in a sample from the patient in comparison to a control.

Likewise, a person skilled in the art will appreciate that the presence of phosporylation at one or more phosphorylation sites of GTPase regulator (RP3) can indicate responsiveness to the FMS-like tyrosine kinase 3 (FLT3) inhibitor, if (preferably statistically significantly) less phosphorylation sites of GTPase regulator (RP3) are not phosphorylated in a sample from the patient in comparison to a control. In other words, a person skilled in the art will appreciate that the presence of phosporylation at one or more phosphorylation sites of GTPase regulator (RP3) can indicate responsiveness to the FMS-like tyrosine kinase 3 (FLT3) inhibitor, if more phosphorylation sites of GTPase regulator (RP3) are phosphorylated in a sample from the patient in comparison to a control.

Without deferring from the gist of the present invention, a proliferative diseased cell can be considered as responsive to an FLT3 inhibitor, if phosphorylated phosphorylation sites of one or more of B-cell lymphoma/leukemia 1 1A protein (BCL1 1 A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), and/or Ran- binding protein 3 (RANBP3), are decreased in comparison to a control; and/or if phosphorylated phosphorylation sites of GTPase regulator (RP3) are increased in comparison to a control.

It is contemplated herein that the phosphorylation status of a plurality of proliferative diseased cells in a sample of a patient is to be determined. As mentioned above, a person skilled in the art will appreciate that proliferative diseased cells in a sample can have a different phosphorylation status. In accordance with the above, the term "absence" or "presence" of phosphorylation can refer to an "decrease" or "increase" of phosphorylated phosphorylation sites in comparison to a control, if, for example, a plurality of proliferative diseased cells in a sample of a patient is to be determined.

For example, a person skilled in the art can perform an absolute determination (i.e. quantification) of phosphorylated proteins of one or more of B-cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) molecules in a sample of a patient and in a control, e.g. a person skilled in the art can perform an absolute determination (i.e. quantification) of phosphorylated phosporylation sites of one or more of B-cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), an-binding protein 3 (RANBP3) and/or GTPase regulator (RP3) in a sample of a patient and in a control. It is envisaged that the phosphorylation status of all or essentially all protein molecules of B-cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3) and/or GTPase regulator (RP3) molecules is determined. Thereby, an increase or decrease of phosphorylation of the proteins as defined above in a patient sample compared to a control can be determined.

For a reliable determination (such as the absoluate determination/quantification) as defined above, it is envisaged that the number of cells in a patient sample and control sample is the same or essentially the same and/or that the quantification is normalized to the total protein content of the sample and the control. Typically, the total protein content is determined before the absolute amount of phosphorylated proteins of one or more of B-cell lymphoma leukemia 11A protein (BCL11A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3) and/or GTPase regulator (RP3) molecules in a sample of a patient and in a control is determined. Routine techniques can be employed in order to determine the total amount of protein, like the Bradford assay and so on. The "total amount of protein" refers to the amount of all proteins/protein molecules in a sample of a patient or a control sample, i.e. the amount of all proteins including the amount of protein molecules of one or more of B-cell lymphoma/leukemia 1 1A protein (BCL1 1A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3) and/or GTPase regulator (RP3) molecules.

Accordingly, in one aspect of the present invention, the herein provided method is a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of one or more of B-cell lymphoma/leukemia 11A protein (BCL1 1A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein a decrease of phosphorylated phosphorylation sites of one or more of B-cell lymphoma/leukemia 1 1A protein (BCLl lA), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), and/or Ran- binding protein 3 (RANBP3), in comparison to a control is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor and/or

wherein an increase of phosphorylated phosphorylation sites of GTPase regulator (RP3) in comparison to a control is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

In a preferred embodiment, the present invention relates to a method of determining whether proliferative diseased cells are responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of B-cell lymphoma/leukemia 11A protein (BCLl lA), in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor,

wherein a decrease of phosphorylated phosphorylation sites of B-cell lymphoma/leukemia 1 1A protein (BCLl lA), in comparison to a control is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether proliferative diseased cells are responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Lamin A/C (LMN1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor,

wherein a decrease of phosphorylated phosphorylation sites of Lamin A/C (LM 1) in comparison to a control is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether proliferative diseased cells are responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl ), in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor,

wherein a decrease of phosphorylated phosphorylation sites of endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPDl), in comparison to a control is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether proliferative diseased cells are responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Ran-binding protein 3 (RANBP3), in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor,

wherein a decrease of phosphorylated phosphorylation sites of Ran-binding protein 3 (RANBP3), in comparison to a control is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether proliferative diseased cells are responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor,

All explanations and definitions provided herein in relation to "proliferative diseased cell(s)", "FMS-like tyrosine kinase 3 (FLT3) inhibitor(s)", "phosphorylation status", "phosphorylation site" "endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1)", "B-cell lymphoma/leukemia 11 A protein (BCL11A)", "Ran-binding protein 3 (RANBP3)", "GTPase regulator (RP3)", "Lamin A/C (LMN1)", "sample", "patient", and the like apply, mutatis mutandis, to the herein above provided aspect of the present invention.

In the aspects of the present invention relating to the phosphorylation status/the phosphosignature, the phosphorylation site can be one or more of the phosphorylation sites SI 60 of endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1), S630 of B- cell lymphoma/leukemia 11A protein (BCL11A), S333 of Ran-binding protein 3 (RANBP3), S96T of GTPase regulator (RP3) and/or S458 of Lamin A/C (LMN1). In other words, the phosphorylation site(s) can be as follows (It is to be understood that any of the aspects of the invention described herein can be combined with any of the following aspects): a. S160 of EEPD1;

b. S630 of BCLl lA;

c. S333 of RANBP3;

d. S961 of RP3; and/or

e. S458 f LMNl .

Also the following phosphorylation sites are predictive (i.e. indicate responsiveness to an FLT3 inhibitor):

S205, S328, S608, S625 and/or S718 of BCL11A; and

S25 and/or S554 of EEPD1.

These phosphorylation sites are also shown in the amino acid sequence of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1) (SEQ ID NO. l), B-cell lymphoma/leukemia 1 1A protein (BCL1 1A) (SEQ ID NO: 2), Ran-binding protein 3 (RANBP3) (SEQ ID NO: 3), GTPase regulator (RP3) (SEQ ID NO. 4) and Lamin A/C (LMN1 ) (SEQ ID NO. 5). It is to be understood that presence or absence of phosphorylation at such phosphorylation sites can be determined by routine techniques in full length proteins (exemplary full length proteins are shown in SEQ ID No. 1-5), fragments of such full length proteins, variants and the like without deferring from the gist of the invention. It is apparent that the term "phosphorylation site" refers to a given amino acid sequence at a given position of the proteins BCL1 1A, EEPD1, L3VIN1, RANBP3 and RP3, respectively.

It was shown herein that the phosphorylation status of further phosphorylation sites of BCLl lA correlates with that of S630 of BCLl lA; see Fig. 8. These phosphorylation sites are S205, S328, S608, S625 and/or S718. Thus, it is plausible that the phosphorylation status of BCL11A as such is indicative of responsiveness to the FMS-like tyrosine kinase 3 (FLT3) inhibitor.

In a preferred embodiment, the present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of B-cell lymphoma/leukemia 11A protein (BCLl lA) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor,

wherein the absence of phosporylation at one or more phosphorylation sites of B-cell lymphoma/leukemia 11A protein (BCLl lA) is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

Furthermore, it was shown herein that the phosphorylation status of further phosphorylation sites of EEPD1 correlates with that of SI 60 of EEPD1; see Fig. 8. These phosphorylation sites are S25 and S554 of EEPD1, Thus, it is plausible that the phosphorylation status of EEPD1 as such is indicative of responsiveness to the FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor,

wherein the absence of phosporylation at one or more phosphorylation site of endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1) is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Ran-binding protein 3 (RANBP3), in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor,

wherein the absence of phosporylation at one or more phosphorylation site of Ran-binding protein 3 (RANBP3) is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor,

wherein the presence of phosporylation at one or more phosphorylation sites of GTPase regulator (RP3) is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Lamin A/C (LMN1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor,

wherein the absence of phosporylation at one or more phosphorylation sites of Lamin A/C (LMN1), is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

As shown in Figure 6, the phosphorylation status of the herein disclosed individual markers is indicative for responsiveness to a FMS-like tyrosine kinase 3 (FLT3) inhibitor. It is demonstrated herein that the phosphorylation status of the herein disclosed phosphorylation sites of the individual markers is indicative for responsiveness to a FMS-like tyrosine kinase 3 (FLT3) inhibitor; such phosphorylation sites are

a. S160 of EEPD1 ;

b. S630 of BCLl lA;

c. S333 of RANBP3;

d. S961 of RP3; or

e. S458 of LMNl.

In a preferred embodiment, the present invention relates to a method of detemiining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of B-cell lymphoma leukemia 11A protein (BCLl lA) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is S630 of BCLl l A.

It was shown herein that the phosphorylation status of further phosphorylation sites of BCLl lA correlates with that of S630 of BCLl lA; see Fig. 8. These phosphorylation sites are S205, S328, S608, S625 and/or S718. Therefore, it is plausible that the phosphorylation status of these phosphorylation sites S205, S328, S608, S625 and/or S718 of BCL11A is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of B-cell lymphoma/leukemia 1 1A protein (BCL1 1A) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is one or more of S630, S205, S328, S608, S625 and/or S718 of BCL1 1 A.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is SI 60 of EEPD1.

Furthermore, it was shown herein that the phosphorylation status of further phosphorylation sites of EEPD1 correlates with that of S160 of EEPD1 ; see Fig. 8. These phosphorylation sites are S25 and S554 of EEPD1. Therefore, it is plausible that the phosphorylation status of these phosphorylation sites S25 and S554 of EEPD1 is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is one or more of SI 60, S25 and/or S554 of EEPD1.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Ran-binding protein 3 (RANBP3), in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is S333 of RANBP3.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is S961 of RP3.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Lamin A/C (LMN1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site S458 of LMN1.

In a preferred embodiment, the present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of B-cell lymphoma/1 eukemia 11A protein (BCL1 1A) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is S630 of BCLl lA,

wherein the absence of phosporylation at said phosphorylation site of B-cell lymphoma/leukemia 11A protein (BCLl lA) is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of B-cell lymphoma/leukemia 11A protein (BCLl lA) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is one or more of S630, S205, S328, S608, S625 and/or S718 of BCLl lA, wherein the absence of phosporylation at said one or more phosphorylation sites of B-cell lymphoma/leukemia 11A protein (BCL11A), is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is SI 60 ofEEPDl,

wherein the absence of phosporylation at said phosphorylation site of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1) is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of endonuclease/exonudease/phosphatase family domain-containing protein 1 (EEPD1 ) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is one or more of S160, S25 and/or S554 ofEEPDl,

wherein the absence of phosporylation at said one or more phosphorylation sites of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Ran-binding protein 3 (RANBP3), in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is S333 of RANBP3,

wherein the absence of phosporylation at said phosphorylation site of Ran-binding protein 3 (RANBP3) is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor. The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site is S961 of RP3,

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Lamin A/C (LMN1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor, wherein said phosphorylation site S458 of LMN1 ,

wherein the absence of phosporylation at one or more phosphorylation sites of Lamin A/C (LMN1) is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

It is envisaged herein that the phosphorylation status (e.g. presence or absence of phosphorylation) of a combination of two, three, four or more of the phosphorylation sites SI 60 of EEPDl ; S630 of BCL11A; S333 of RANBP3; S961 of RP3; and S458 of LMN1 can be determined. Any combination of two, three, four or more of the phosphorylation sites S160 of EEPDl ; S630 of BCL11A; S333 of RANBP3; S961 of RP3; and S458 of LMN1 is envisaged herein.

S160 of EEPDl and S630 of BCL11A;

S160 of EEPDl and S333 of RANBP3;

S160 of EEPDl and S961 of RP3;

S160 of EEPDl and S458 of LMN1 ;

S160 of EEPDl, S630 of BCL1 1A and S333 of RANBP3;

S160 of EEPDl, S630 of BCL11 A and S961 of RP3;

S160 of EEPDl, S630 of BCL11A and S458 of LMN1;

S160 of EEPDl, S333 of RANBP3, and S961 of RP3;

S160 of EEPDl, S333 of RANBP3, and S458 of LMN1; S160 of EEPD1, S961 of RP3 and S458 of LMN1 ;

S630 of BCL11 A and S333 of RANBP3;

S630 of BCL1 1 A, and S961 of RP3;

S630 of BCL1 1A and S458 of LM 1 ;

S630 of BCL11A, S333 of RANBP3, and S961 of RP3;

S630 of BCL1 1A, S333 of RANBP3, and S458 of LMN1 ;

S630 ofBCLl lA, S961 of RP3 and S458 of LMN1;

S333 of RANBP3 and S961 of RP3;

S333 of RANBP3 and S458 of LMN1 ;

S333 of RANBP3, S961 of RP3 and S458 of LMN1 ; or

S961 of RP3 and S458 of LMN1 ;

and any combination thereof.

The meaning of the terms "cell(s)", "tissue(s)" and "cell culture(s)" is well known in the art and may, for example, be deduced from "The Cell" (Garland Publishing, Inc., third edition). Generally, the term "cell(s)" used herein refers to a single cell or a plurality of cells. The term "plurality of cells" means in the context of the present invention a group of cells comprising more than a single cell. Thereby, the cells out of said group of cells may have a similar function. Said cells may be connected cells and/or separate cells. The term "tissue" in the context of the present invention particularly means a group of cells that perform a similar function. Preferably, the cell(s)/ tissue(s) that are to be determined to respond to an FLT3 inhibitor comprise/are derived from or are (a) proliferative diseased cell(s) as defined herein. The cells may, for example, be obtained from a bone marrow sample or a blood sample (like a peripheral blood sample), in particular (a) bone marrow sample(s) or (a) blood sample(s) from a patient/subject suffering from neoplasia. The cells may, for example, be obtained from (a) bone marrow sample(s) or (a) blood sample(s) (like a peripheral blood sample) from a patient/subject being prone to suffer from neoplasia or from a patient/subject suspected to suffer from neoplasia. It is preferred herein that said patient/subject is a human. The sample may be a biopsy, e.g. an aspirate. Thus, the method for determinining the responsiveness of (a) proliferative diseased cell(s) can be used to determine whether a patient/subject/individual suffering from neoplasia, suspected to suffer from neoplasia or being prone to suffer from neoplasia is responsive to an FLT3 inhibitor.

Preferably, the sample as defined and to be used herein is generally a bone marrow sample. If a blood sample (like a peripheral blood sample) is to be used herein, it is preferred that such a blood sample is to be used for determining the phosphorylation status of BCL11 A, EEPD1 and/or LMN1 as defined herein above. BCL11A, EEPD1 and/or LM 1 show a strong correlation in their phosphorylation in the respective bone marrow and peripheral blood samples (Fig. 7).

For example, for validation purposes in animal experiments, the patient/subject suffering from neoplasia, being prone to suffer from neoplasia, suspected to suffer from neoplasia can be a non- human mammal. Thus, the proliferative diseased cell(s) can be determined in a sample of such a mammal. The meaning of the term "mammal" is well known in the art and can, for example, be deduced from Weh er und Gehring (1995; Thleme Verlag). Non-limiting examples for mammals are even-toed ungulates such as sheep, cattle and pig, odd-toed angulates such as horses as well as carnivors such as cats and dogs. In the context of this invention, it is particularly envisaged that samples are derived from organisms that are economically, agronomically or scientifically important. Scientifically or experimentally important organisms include, but are not limited to, mice, rats, rabbits, guinea pigs and pigs.

The term "sample" as used herein relates, inter alia, to a biological sample, including but not limiting to tissue samples or samples comprising said (tumor or cancer) cell(s) to be tested and/or scrutinized. As used here, the terms "sample" or "sample to be evaluated/measured/tested/scrutinized/assessed" may also comprise tissue from biopsies etc. The term "sample" is preferably an in vitro sample. The definition of "control" or "contol samples" was provided herein above and applies, mutatis mutantis, to the the embodiments of the invention provided herein. The herein provided method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase (FLT3) inhibitor is preferably an in vitro method.

The term "responder" as used herein can refer to patients with complete remission (CR), complete remission with incomplete haematological recovery (CRi), complete remission with incomplete platelate recovery (CRp), and partial remission (PR). Patients with stable disease (SD) or no response can be seen as ,,ηοη-responder".

Whether a patient determined to respond before or during the FLT3 inhibitor therapy does indeed respond to an FLT3 inhibitor can be determined according to the above classification that is known in the art (complete remission (CR), complete remission with incomplete haematological recovery (CRi), complete remission with incomplete platelate recovery (CRp), and partial remission (PR)). For example, the number of proliferative diseased cells in a sample from a patient prior to start of treatment with an FLT3 inhibitor, optionally in a sample from a patient during the treatment with an FLT3 inhibitor and in a sample from a patient after termination of the treatment with an FLT3 inhibitor can be determined to confirm that the patient did indeed respond to the FLT3 inhibitor. A decrease of proliferative diseased cells in a sample from a patient during or after termination of the treatment with an FLT3 inhibitor compared to the initial number of proliferative diseased cells (i.e. the number of cells in a sample from a patient prior to start of treatment with an FLT3 inhibitor) can be used to confirm that the patient did indeed respond to the FLT3 inhibitor. Accordingly, the herein provided markers are primarily useful in predicting a response to an FLT3 inhbitor (e.g. the response is determined before the first cycle of the therapy/treatment started). The markers are also useful for monitoring a response during therapy/treatment with an FLT3 inhbitor.

The herein provided methods can further comprise determining the expression level of Lamin A/C (LM 1). A decrease in said expression level in comparison to the control can be indicative of the responsiveness to the FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The expression level of Lamin A/C (LMN1) is, independently of the phosphorylation status/phosphosignature of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), B-cell lymphoma/leukemi 1 1A protein (BCL11A), Ran-binding protein 3 (RANBP3), GTPase regulator (RP3) and Lamin A/C (LMNl), indicative of responsiveness to a FLT3 inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the expression level of Lamin A C (LMNl ) in a sample of a patient, wherein said expression level is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor. A decrease in said expression level in comparison to the control can be indicative of the responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The expression level of Lamin A/C (LMNl ) is preferably at least 2.5-fold, more preferably at least 5-fold decreased in comparison to the control. The expression level of Lamin A/C (LMN1) can be the mRNA expression level of Lamin A/C (LMN1). The mRNA expression level can be assessed by in situ hybridization, micro-arrays, or RealTime PCR and the like.

If the gene product is an RNA, in particular an mRNA (e.g. unspliced, partially spliced or spliced mRNA), determination can be performed by taking advantage of northern blotting techniques, in situ hybridization, hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for mRNA transcripts or PCR techniques, like, quantitative PCR techniques, such as Real time PCR. These and other suitable methods for binding (specific) mRNA are well known in the art and are, for example, described in Sambrook and Russell (2001 , loc. cii). A skilled person is capable of determining the amount of the component, in particular said gene products, by taking advantage of a correlation, preferably a linear correlation, between the intensity of a detection signal and the amount of the gene product to be determined.

The expression level of Lamin A/C (LMN1) can be the protein expression level of Lamin A/C (LMN1). The protein expression level can be assessed by immunoassay, gel- or blot-based methods, IHC, mass spectrometry, flow cytometry, FACS or Western blotting techniques (or the like). Quantification of the protein expression level can accordingly be perfonried by taking advantage of well known techniques. Generally, a person skilled in the art is aware of methods for the quantitation of (a) polypeptide(s)/protein(s). Amounts of purified polypeptide in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular polypeptide in a mixture may rely on specific binding, e.g. of antibodies. Antibodies to be used for quantification and detection of the expression of Lamin A/C (LMN1) are well known and commercially available exemplary anti- LMN1 antibodies are poly-clonal rabbit antibodies HPA006660 (Atlas Antibodies, Stockholm, Sweden), and #2032 (Cell Signaling, Danvers, Massachusetts).

Such antibodies may be used in the herein provided detection and quantitation methods. Specific detection and quantitation methods exploiting the specificity of antibodies comprise for example immunohistochemistry (in situ). Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies. Electrophoresis may be multi-dimensional such as 2D electrophoresis. Usually, polypeptides are separated in 2D electrophoresis by their apparent molecular weight along one dimension and by their isoelectric point along the other direction. Alternatively, protein quantitation methods may involve but are not limited to mass spectrometry or enzyme-linked immunosorbant assay methods.

Further, it was demonstrated herein that the phosphorylation status of further phosphorylation sites of BCL1 1A correlates with that of S630 of BCLl 1 A; see Fig. 8. It is therefore plausible that the phosphorylation status correlates in turn with the expression level and that the responder and non- responder differ in relation to the expression level of BCLl 1 A. This suggests in turn that the expression of BCLl 1 A can be used in the alternative/independently or in addition to its phosphorylation status to determine whether a proliferative diseased cell or patient is responsive to an FLT3 inhibitor.

The expression of BCLl 1 A can be used in addition to/in combination with the phosphorylation status of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), B-cell lymphoma/leukemia 1 1A protein (BCLl 1 A), Ran-binding protein 3 (RANBP3), GTPase regulator (RP3) and/or Lamin AJC (LMN1) to determine whether a proliferative diseased cell is responsive to an FLT3 inhibitor. The expression of BCLl 1 A can be used in addition to/in combination with the expression level of Lamin A/C (LMN1 ) to determine whether a proliferative diseased cell is responsive to an FLT3 inhibitor.

Thus, the herein provided methods can further comprise determining the expression level of BCLl 1 A. A decrease in said expression level in comparison to the control can be indicative of the responsiveness to the FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to a method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the expression level of BCLl 1 A in a sample of a patient, wherein said expression level is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor. A decrease in said expression level in comparison to the control can be indicative of the responsiveness to said FMS- like tyrosine kinase 3 (FLT3) inhibitor.

The expression level of BCLl 1A is preferably at least 2.5-fold, more preferably at least 5-fold decreased in comparison to the control. The expression level of BCL11A can be the mRNA expression level of BCL11A. The mRNA expression level can be assessed by in situ hybridization, micro-arrays, or RealTime PCR and the like.

The expression level of BCL1 1A can be the protein expression level of BCL11A. The protein expression level can be assessed by immunoassay, gel- or blot-based methods, IHC, mass spectrometry, flow cytometry, FACS or Western blotting techniques (or the like). The explanations provided herein above in relation to the (protein) expression level of Lamin A C (LMNl) apply, mutatis mutandis, here.

As used in context of the methods of the present invention, a non-limiting example of a "control" is preferably a "non-responder" control, for example a sample/cell/tissue obtained from one or more healthy subjects or one or more patients that suffer from a neoplasia (like leukemia, such as AML) and are known to be not responsive to an FLT3 inhibitor. Another example for a "non-responder" control is a cell line/sample/cell/tissue that shows no response to an FLT3 inhibitor in an ex-vivo test. Another non-limiting example of a "control" is an "internal standard", for example purified or synthetically produced proteins and/or peptides or a mixture thereof, where the amount of each protein/peptide is gauged by using the "non-responder" control described above. In particular, this "internal standard" can contain phosphorylated peptides (e.g. fragments of the respective full length proteins comprising the herein described phosphorylation sites) of one or more of B-cell lymphoma/1 eukemia 1 1A protein (BCL11A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3). In addition, or independently, this "internal standard" can contain the protein Lamin A/C (LMN1) and/or B-cell lymphoma/leukemia 11A protein (BCL11 A).

A further non-limiting example of a "control" may be a "healthy" control, for example a sample/cell/tissue obtained from a healthy subject or patient that is not suffering from a neoplasia (like leukemia, such as AML) or a cell obtained from such a subject. In accordance with the above, the reference or control status e.g. of one or more of B-cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) is that determined in (a sample of) the corresponding healthy control subject/patient, i.e. it is the "normal" status of one or more of B-cell lymphoma/leukemia 11A protein (BCL1 1A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), , Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3).

The control may also be a sample/cell/tissue obtained from the individual or patient suspected of suffering from the neoplasia provided that the sample/cell/tissue does not contain proliferative diseased cells as defined herein. In a further alternative, the "control" may be a sample/cell/tissue obtained from an individual or patient suffering from the neoplasia (like leukemia, such as AML), that has been obtained prior to the development or diagnosis of said neoplasia.

The phosphorylation can be detected by routine techniques, such as immunoassays, IHC, mass spectrometry or intracellular flow cytometry.

As explained above, the phosphorylation status of one or more of B-cell lymphoma/leukemia 11A protein (BCLl lA), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) indicates, independently of the expression level of Lamin A/C (LMN1) and/or the expression level of B-cell lymphoma/leukemia 11 A protein (BCLl lA), whether a cell or individual/patient is responsive to an FLT3 inhibitor. Accordingly, it is also envisaged herein that the phosphorylation status of one or more of B-cell lymphoma/leukemia 11A protein (BCLl lA), Lamin A C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) can be evaluated independently of the expression level of Lamin A C (LM 1) and/or the expression level of B-cell lymphoma/leukemia 11A protein (BCLl lA). The present invention, therefore, also provides for the assessment/elucidation/scrutinization of the phosphosignature of one or more of B-cell lymphoma/leukemia 11A protein (BCLl lA), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in determining the responsiveness of a proliferative diseased cell to an FLT3 inhibitor. This assessment/elucidation/scrutinization of the phosphosignature of one or more of B-cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1 ), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) may be carried out individually or may be carried out in combination with the assessment/elucidation/scrutinization of the expression level of Lamin A/C (LMN1) and/or the expression level of B-cell lymphom a/leukemia 11A protein (BCL11A). Said expression level may be the expression level of Lamin A/C mRNA or Lamin A/C protein. Said expression level may be the expression level of BCL11A mRNA or BCL1 1A protein. Ample details are provided herein and the person skilled in the art is readily in a position to carry out the invention as described. In this context it is also of note that when the assessment/elucidation/scrutinization of the phosphosignature of one or more of B-cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) is combined with a concomitant assessment elucidation/scrutinization of the expression level of Lamin A/C (LMN1) and/or the expression level of B-cell lymphoma/leukemia 11A protein (BCL11A), said assessment/elucidation/scrutinization may take place on the same (celluar) sample or on different samples. The assessment/elucidation scrutinization may take place at the same time or on different time points.

Again, when a concomitant assessment/elucidation/scrutinization takes place, the elucidation of the expression level of Lamin A/C (LMN1) and/or the expression level of B-cell lymphoma/leukemia 11A protein (BCL1 1 A) may be the confirmation of the results obtained when the "phosphosignature" of one or more of B-cell lymphoma/leukemia 1 1A protein (BCL1 1A), Lamin A C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) is assessed and, vice versa, the elucidation of the "phosphosignature" may be the confirmation of the expression level(s).

The following relates to FLT3 inhibitors to be used in accordance with the present invention.

As used herein, a "FLT3 inhibitor" or "inhibitor of FLT3" (and the like) refers to any compound capable of (at least partially) downregulating, decreasing, suppressing or otlierwise regulating the amount, expression and/or activity of FLT3. FLT3 is known in the art. Corresponding sequences can be retrieved from databases like NCBI and an exemplary sequence is also provided herein (SEQ ID NO: 7). Inhibition of FLT3 can be achieved by any of a variety of mechanisms known in the art, including, but not limited to binding directly to the FLT3 polypeptide, denaturing or otherwise inactivating FLT3, or inhibiting the expression of the FLT3 gene (e.g., transcription to mRNA, translation to a nascent polypeptide, and/or final polypeptide modifications to a mature protein), which encodes the FLT3 protein. Generally, FLT3 inhibitors may be proteins, polypeptides, nucleic acids, small molecules, or other chemical moieties. The term "inhibitor of FLT3" means accordingly in this context a compound capable of inhibiting the expression and/or activity of a FLT3 kinase as defined herein. An "inhibitor of aFLT3 kinase" may, for example, interfere with transcription of a gene encoding FLT3, processing (e.g. splicing, export from the nucleus and the like) of the gene product (e.g. unspliced or partially spliced mRNA) and/or translation of the gene product (e.g. mature mRNA). The inhibitor of a FLT3 kinase may also interfere with further modification (like phosphorylation) of the FLT3 polypeptide/protein encoded by the FLT3 gene and thus completely or partially inhibit the activity of FLT3. Furthermore, the inhibitor of FLT3 may interfere with interactions of FLT3 with other proteins. It is envisaged that FLT3 inhibitors to be used in accordance with the invention show a high potency (demonstrated by a low IC50 value) for inhibiting FLT3 activity. The FLT3 inhibitor may be a selective FMS-like tyrosine kinase 3 (FLT3) inhibitor.

Exemplary FLT3 inhibitors to be used herein are quizartinib (AC220), crenolanib (CP-868596), midostaurin (PKC-412), lestaurtinib (CEP-701), 4SC-203, TTT-3002, sorafenib (Bay-43-0006), Ponatinib (AP-24534), sunitinib (SU-11248), and/or tandutinib (MLN-0518), or (a) pharmaceutically acceptable salt(s), solvate(s), and/or hydrate(s) thereof. Preferably, the FMS-like tyrosine kinase 3 (FLT3) inhibitor is quizartinib (AC220) or pharmaceutically acceptable salt(s), solvate(s), and/or hydrate(s) thereof.

These and further exemplary inhibitors to be used herein are described in more detail below.

Brand Name: Quizartinib

Code Name: AC-220

Structure:

lUPAC Name: N-(5-tert-Butylisoxazol-3-yl)-N'-[4-[7-[2-(4-morpholinyl)ethoxy]imidazo[2,l- b][l,3]benzothiazol-2-yl]phenyl]urea

Affinities: FLT3 (1.6nM), KIT (4.8nM), PDGFRB (7.7nM), RET (9.9nM)₅ PDGFRA (1 InM),

CSF1R (12nM) (Zarrinkar, Gunawardane et al. 2009)

Clinical Phase: Phase II (AML)

Developer: Ambit Biosciences (Originiator), Astellas Pharma

AC-220 is described for example in Zarrinkar, P. P., R. N. Gunawardane, et al. (2009). "AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML)." BJood 114(14): 2984-2992.

Brand Name: Crenolanib

Code Name: CP-868596

Structure:

IUPAC Name: 1 -(2-[5-[(3-Methyloxetan-3-yl)methoxy]- 1 H-benzimidazol-1 -yl]quinolin-8- yl)piperidin-4-amine benzenesulfonate

Affinities: FLT3, PDGFRb (Galanis, Blood. 2013.)

Clinical Phase: Phase II (AML)

Developer: AROG Pharmaceuticals, Pfizer (originator), Astellas Phara (originator) Brand Name: Midostaurin

Code Name: PKC-412

Structure:

IUPAC Name: N-[(9S,10R,1 lR,13R)-10-Methoxy-9-methyl-l -oxo-9,13-epoxy-2,3, 10,11, 12,13- eximydro-l H^H-diindoloCl^-gh^^^^^

1 l-yl]-N-methylbenzamide

Affinities: PKNl (9.3nM), TBKl (9.3nM), FLT3 (1 InM), JAK3 (12nM), MLKl (15nM), and

30 targets in the range 15-1 lOnM (Zarrinkar, Gunawardane et al. 2009, loc. cit.)

Clinical Phase: Phase III (AML)

Developer: Novartis

Brand Name: Lestaurtinib

Code Name: CEP-701

IUPAC Name: 9(S),12fR)-Epoxy-10(S)- ydroxy-10-(hydroxymethyl)-9-methyl-2,3,9,10,l 1,12- hexahydrodiindolo[ 1 ,2,3-fg:3 2 1 '-kl]pyrrolo[3 ,4-i] [ 1 ,6]benzodiazocin- 1 -one

Affinities: FLT3 (Levis, Blood. 2002;99(1 1 ):3885- 1.), TRKA, TRKB, TRKC (George,

Cancer Res. 1999;59( ] 0):2395-401 ).

Clinical Phase: Phase 11 (AML)

Developer: Cephalon, Kyowa Hakko (Originator)

Brand Name: 4SC-203

Code Name: 4SC-203

Structure:

IUPAC Name: N-(2-Methoxy-5-metliylphenyl)-N'-[6-[6-methoxy-7-[3-(4-methylpiperazin- 1 - yl)propoxy]quinazolin-4-ylamino]benzothiazol~2-yl]urea

Affinities: FLT3, VEGFR

Clinical Phase: Phase I (AML)

Developer: 4SC (originator), ProQinase Code Name: TTT-3002

Structure:

Affinities: FLT3 (Wall, Blood (ASH Annual Meeting Abstracts). 2012; 120:866);

LRRK2 (Yao, Human molecular genetics. 2013;22(2):328-44).

Clinical Phase: Preclinical

Developer: Tautatis (originator)

Brand Name: Sorafenib

Code Name: Bay-43-0006

Structure:

IUPAC Name: 4-[4-[3-[4-Chloro-3-(trifluoromethyl)phenyl]ureido]phenoxy]-N-methylpyridine-2- carboxamide

Affinities: DDRl (1.5nM), HIPK4 (3nM)₅ ZAK (6nM), DDR2 (7nM), FLT3 (13nM), and 15 targets in the range 13-130nM (Zarrinkar, Gunawardane et al. 2009, loc. cit.) Clinical Phase: Launched (renal and heptacellular carcinoma), Phase I/O (blood cancer) Developer: Bayer

Brand Name: Ponatinib

Code Name: AP-24534 Structure:

IUPAC Name: 3-[2-(Imidazo[l,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-[4-(4-methylpiperazin-l- ylmethyl)-3-(trifluoromethyl)phenyl]benzamide

Affinities: BCR-ABL, FLT3, KIT, FGFR1, PDGFRa (Gozgit, Mol Cancer Ther.

201 1 ;10(6):1028-35).

Clinical Phase: Phase II (AML)

Developer: Ariad Pharmaceuticals (originator)

Brand Name: Sunitinib

Code Name: SU-11248

Structure:

lUPAC Name: (Z)-N-[2-(Diethylamino)ethyl]-5-(5-fluoro-2-oxo-2,3-dihydro-lH-indol-3- ylidenemethyl)-2,4-dimethyl-lH-pyrrole-3-carboxamide 2(S)~hydroxybutanedioic acid (1 :1) N-[2-(Diethylamino)ethyl]-5-[(Z)-(5-fluoro-2-oxo-l,2-dihydro-3H-indol- 3 -ylidene)methyl] -2,4-dimethyl- 1 H -pyrrole-3 -carboxamide L-malate

Affinities: PDGFRB (0.075nM), KIT (0.37nM), FLT3 (0.47nM), PDGFRA (0.79nM),

DRAK1 (l.OnM), VEGFR2 (1.5nM), FLT1 (1.8nM), CSF1R (2.0nM) (Zarrinkar, Gunawardane et al. 2009, loc. cit.)

Clinical Phase: Launched (renal cell carcinoma, gastrointestinal stromal cancer, neuroendocrine pancreas), phase I (AML)

Developer: Pfizer (Originator)

Brand Name: Tandutinib

Code Name: MLN-0518

Structure:

IUP AC Name: N-(4-Isopropoxyphenyl)-4-[6-methoxy-7-[3-(l -piperidinyl)propoxy]quinazolin-4- yl]piperazine- 1 -carboxamide

Affinities: PDGFRA (2.4nM), KIT (2.7nM), FLT3 (3nM), PDGFRB (4.5nM), CSF1R

(4.9nM) (Zarrinkar, Gunawardane et al. 2009, loc. cit.)

Clinical Phase: discontinued

Developer: Kyowa Hakko Kirin (Originator), Millennium Pharmaceuticals (Originator),

National Cancer Institute, Takeda (Originator) FLT3 inhibitors to be used in accordance with the present invention are not limited to the herein described or further known exemplary inhibitors. Accordingly, also further inhibitors or even yet unknown inhibitors may be used in accordance with the present invention. Such inliibitors may be identified by the methods described and provided herein and methods known in the art, like high- throughput screening using biochemical assays for inhibition of FLT3.

Assays for screening potential FLT3 inhibitors and, in particular, for identifying FLT3 inhibitors as defined herein, comprise, for example, in vitro competition binding assays to quantitatively measure interactions between test compounds and recombinantly expressed kinases¹ (Fabian et al; Nat Biotechnol. 2005 23(3):329-36). Hereby, competition with immobilized capture compounds and free test compounds is performed. Test compounds that bind the kinase active site will reduce the amount of kinase captured on solid support, whereas test molecules that do not bind the kinase have no effect on the amount of kinase captured on the solid support. Furthermore, inhibitor selectivity can also be assessed in parallel enzymatic assays for a set of recombinant protein kinases.^2,3 (Davies et al. ;, Biochem. J. 2000 351 : 95-105; Bain et al. Biochem. J. 2003 371 : 199-204). These assays are based on the measurement of the inhibitory effect of a kinase inhibitor and determine the concentration of compound required for 50% inhibition of the protein kinases of interest. Proteomics methods are also an efficient tool to identify cellular targets of kinase inliibitors. Kinases are enriched from cellular lysates by immobilized capture compounds, so the native target spectrum of a kinase inhibitor can be determined.⁴ (Godl _et al;. Proc Natl Acad Sci USA. 2003 100(26):! 5434-9).

Assays for screening of potential inhibitors and, in particular, for identifying inhibitors as defined herein, are, for example, described in the following papers:

Fabian et al; Nat Biotechnol. 2005 23(3):329-36

Davies et al. ;, Biochem. J. 2000 351 : 95-105.

Bain et al. ; , Biochem. J. 2003 371 : 199-204.

Godl et al;. Proc Natl Acad Sci USA. 2003 100(26): 15434-9.

The above papers are incorporated herein in their entirety by reference.

Based on his general knowledge a person skilled in the art is in the position to identify inhibitors or verify the inhibiting activity of compounds suspected of being inhibitors. These tests may be employed on appropriate cell(s)/ tissue(s)/cell culture(s) or cell(s)/ tissue(s)/cell culture(s) derived from bone marrow samples or blood samples (like a peripheral blood sample).

It is also envisaged herein that responsiveness to two or more different FLT3 inhibitors (i.e. inhibitors having different chemical formulae, optionally non-structurally related inhibitors) may be determined simultaneously. It is envisaged herein that responsiveness to only one inhibitor is tested at one time. FLT3 inhibitors to be used and tested in the present invention are described herein.

The following relates to neoplasia(s) as used herein.

The patient whose responsiveness to FLT3 inhibitor(s) is to be determined can be suspected to suffer from neoplasia, is suffering from neoplasia or is being prone to suffer from neoplasia. Likewise, the sample to be assessed can be (obtained) from a patient suspected to suffer from neoplasia, is suffering from neoplasia or is being prone to suffer from neoplasia.

The neoplasia can be a malignant neoplasia, e.g. the patient may show a hematological neoplasm. The malignant neoplasia can be leukemia, like myeloid leukemia or lymphoid leukemia. The myeloid leukemia may be acute myeloid leukemia (AML). Acute myeloid leukemia (AML) is preferred herein. The lymphoid leukemia may be acute lymphoid leukemia (ALL).

The neoplasia may be a myelodysplastic syndrome. Such a myelodysplastic syndrome may be refractory anemia with excess of blasts (RAEB I or RAEB II). The neoplasia may also be a lymphoma, such as Hodgkin lymphoma and non-Hodgkin lymphoma. Samples from patients suffering from lymphomas can be biopsies, such as biopsies of lymph nodes.

In one aspectof the present invention, the proliferative diseased cell(s) or the (sample from the) patient(s) is/are characterized by mutations of FLT3, like activating FLT3 mutations. In other words, mutations of FLT3, like activating FLT3 mutations, are present in the proliferative diseased cell(s) or the (sample from the) patient(s). FLT3 mutations and techniques for determining the presence of FLT3 mutations in a sample of a patient are well known in the art and can be used in accordance with the present invention. For example, mutations of FLT3, like activating FLT3 mutations, are described in the following papers which are incorporated herein by reference: • FLT3-ITD: Nakao ML, Yokota S., Iwai T., aneko H., Horiike S., Kashima K., Sonoda Y., Fujimoto T., Misawa S.: "Internal tandem duplication of the flt3 gene found in acute myeloid leukemia.", Leukemia 10:1911-1918(1996)

• Asp835: Abu-Duhier F.M., Goodeve A.C., Wilson G.A., Care R.S., Peake I.R., Reilly J.T.:

"Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia.", Br. J. Haematol. 113:983-988(2001)

• Ile836: Taketani T., Taki T., Sugita ., Furuichi Y., Ishii E., Hanada R., Tsuchida M., Sugita ., Ida K., Hayashi Y.: "FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with

hyperdiploidy", Blood 103:1085-1088(2004)

The herein provided methods of detennining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor can comprise a step of administering an FMS-like tyrosine kinase 3 (FLT3) inhibitor to the patient.

Furthermore, herein contemplated are in vitro uses of the herein provided markers for detemiining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor.

The present invention relates to an in vitro use of the phosphorylation status of one or more of B- cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) for determining whether a proliferative diseased cell in a sample of a patient is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor.

All explanations and definitions provided herein in relation to "proliferative diseased cell(s)", "FMS-like tyrosine kinase 3 (FLT3) inhibitor(s)", "phosphorylation status", "phosphorylation site" "endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl)", "B-cell lymphoma/leukemia 11A protein (BCL11A)" "Ran-binding protein 3 (RANBP3)", "GTPase regulator (RP3)", "Lamin A/C (LM 1)", "sample", "patient", and the like apply, mutatis mutandis, to the herein above provided aspect of the present invention. The present invention relates to a method of treating a patient, said method comprising selecting a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is determined (i) to have one or more of B-cell lymphoma leukemia 11A protein (BCL1 1A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPDl), and/or Ran-binding protein 3 (RANBP3), not phosphorylated; and/or

fii) to have GTPase regulator (RP3) phosphorylated; and

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient

(i) is not phosphorylated at one or more phosphorylation sites of one or more of B-cell lymphoma/leukemia 1 1A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), and/or Ran-binding protein 3 (RANBP3); and/or

(ii) is phosphorylated at one or more phosphorylation sites of GTPase regulator (RP3).

(i) is not phosphorylated at one or more phosphorylation sites of one or more of B-cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), and/or Ran-binding protein 3 (RANBP3); and/or

(ii) is phosphorylated at one or more phosphorylation sites of GTPase regulator (RP3), wherein the treatment comprises determining the phosphorylation status at one or more phosphorylation sites of one or more of B-cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3) and/or GTPase regulator (RP3) in a sample of a patient.

As mentioned above, explanations and definitions provided herein in relation to "proliferative diseased cell(s)", "FMS-like tyrosine kinase 3 (FLT3) inhibitor(s)", "phosphorylation status", "phosphorylation site" "endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl)", "B-cell lymphoma/leukemia 1 1A protein (BCL11A)", "Ran-binding protein 3 (RANBP3)", "GTPase regulator (RP3)", "Lamin A/C (LMNl)", "sample", "patient", and the like apply, mutatis mutandis, to the herein provided "method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor", "method of treating a patient" and "FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient".

In a preferred embodiment, the present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or morephosphorylation sites of B-cell lymphorna/leukemia 11A protein (BCL1 1A)

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of B-cell lymphorna/leukemia 11A protein (BCL11A),

wherein the treatment comprises determining the phosphorylation status at one or more phosphorylation sites of B-cell lymphorna leukemia 1 1A protein (BCLUA ) in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of Lamin A/C (LMNl ).

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of Lamin A/C (LMNl), wherein the treatment comprises determining the phosphorylation status at one or more phosphorylation sites of Lamin A/C (LMNl) in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1 ). The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1),

wherein the treatment comprises determining the phosphorylation status at one or more phosphorylation sites of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1) in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of Ran-binding protein 3 (RANBP3).

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of Ran-binding protein 3 (RANBP3), wherein the treatment comprises determining the phosphorylation status at a phosphorylation site of Ran-binding protein 3 (RANBP3) in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is phosphorylated at one or more phosphorylation sites of GTPase regulator (RP3).

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is phosphorylated at one or more phosphorylation sites of GTPase regulator (RP3),

wherein the treatment comprises determining the phosphorylation status at one or more phosphorylation sites of GTPase regulator (RP3) in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at a phosphorylation site of endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1), wherein said phosphorylation site is SI 60 of EEPD1. The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at a phosphorylation site of endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPDl), wherein said phosphorylation site is SI 60 of EEPDl,

wherein the treatment comprises determining the phosphorylation status at said phosphorylation site of EEPDl in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), wherein said phosphorylation site is one or more of S160, S25 and/or S554 of EEPDl .

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), wherein said phosphorylation site is one or more of S 160, S25 and/or S554 of EEPDl .,

wherein the treatment comprises determining the phosphorylation status at one or more of said phosphorylation site SI 60, S25 and/or S554 of EEPDl in a sample of a patient.

In a preferred embodiment, the present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at a phosphorylation site of B-cell lymphoma/leukemia 11 A protein (BCL 11 A),

wherein said phosphorylation site is S630 of BCL11 A.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at a phosphorylation site of B-cell lymphoma/leukemia 11 A protein (BCL11 A), wherein said phosphorylation site is S630 of BCL11 A,

wherein the treatment comprises determining the phosphorylation status at said phosphorylation site of BCL11A in a sample of a patient. The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of B-cell lymphoma/1 eukemia 1 1 A protein (BCL1 1A),

wherein said phosphorylation site is one or more of S630, S205, S328, S608, S625 and/or S718 of BCL1 1A.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of B-cell lymphoma/leukemia 1 1A protein (BCL1 1A),

wherein said phosphorylation site is one or more of S630, S205, S328, S608, S625 and/or S718 of BCL11A,

wherein the treatment comprises determining the phosphorylation status at one or more of said phosphorylation site S630, S205, S328, S608, S625 and/or S718 of BCL11A in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at a phosphorylation site of Lamin A/C (LMN1), wherein said phosphorylation site is S458 of LMNL

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at a phosphorylation site of Lamin A/C (LMN1), wherein said phosphorylation site is S458 of LMNl,

wherein the treatment comprises determining the phosphorylation status at said phosphorylation site of LMN1 in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at a phosphorylation site of Ran-binding protein 3 (RANBP3), wherein said phosphorylation site is S333 of RANBP3.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at a phosphorylation site of Ran-binding protein 3 (RANBP3), wherein said phosphorylation site is S333 of RANBP3,

wherein the treatment comprises determining the phosphorylation status at said phosphorylation site of RANBP3 in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is phosphorylated at a phosphorylation site of GTPase regulator (RP3), wherein said phosphorylation site is S961 of RP3.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is phosphorylated at a phosphorylation site of GTPase regulator (RP3), wherein said phosphorylation site is S961 of RP3,

wherein the treatment comprises determining the phosphorylation status at a phosphorylation site of said phosphorylation site of RP3 in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of B-cell lymphoma/leukemia 1 1A protein (BCL11 A), and wherein the expression level of BCL11 A is decreased in comparison to the control.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of B-cell lymphoma/leukemia 1 1A protein (BCLl 1 A), and wherein the expression level of BCL11 A is decreased in comparison to the control, wherein the treatment comprises determining the phosphorylation status at one or more phosphorylation sites of B-cell lymphoma/leukemia 1 1A protein (BCLl 1 A), in a sample of a patient,and wherein the treatment comprises determining the expression of BCLl 1 A in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of B-cell lymphoma/leukemia 11 A protein (BCLl 1 A),

wherein said phosphorylation site is one or more of S630, S205, S328, S608, S625 and/or S718 of BCLl 1 A, and wherein the expression level of BCLl 1 A is decreased in comparison to the control.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is not phosphorylated at one or more phosphorylation sites of B-cell lymphoma/leukemia 11A protein (BCLl 1 A),

wherein said phosphorylation site is one or more of S630, S205, S328, S608, S625 and/or S718 of BCLl 1 A, and wherein the expression level of BCLl 1 A is decreased in comparison to the control, wherein the treatment comprises determining the phosphorylation status at one or more of said phosphorylation sites S630, S205, S328, S608, S625 and/or S718 of BCL11A in a sample of a patient, and wherein the treatment comprises determining the expression of BCLl 1 A in a sample of a patient.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient has a decreased expression level of BCLl 1A in comparison to the control.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient has a decreased expression level of BCLl 1 A in comparison to the control,

wherein the treatment comprises determining the expression of BCLl 1 A in a sample of a patient.

The expression level of BCLl 1 A is preferably at least 2.5-fold, more preferably at least 5-fold decreased in comparison to the control. The expression level of BCLl lA can be the mRNA expression level of BCL11A. The mRNA expression level can be assessed by in situ hybridization, micro-arrays, or RealTime PCR and the like.

The expression level of BCLl lA can be the protein expression level of BCL lA. The protein expression level can be assessed by immunoassay, gel- or blot-based methods, IHC, mass spectrometry, flow cytometry, FACS or Western blotting techniques (or the like). The explanations provided herein above in relation to the (protein) expression level of Lamin A/C (LM l) apply, mutatis mutandis, here.

The present invention relates to the use of a nucleic acid or antibody capable of detecting the phosphorylation status of one or more of B-cell lymphoma/leukemia 1 1A protein (BCLl lA), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) . Also provided is the use of a nucleic acid or antibody capable of detecting the expression level of Lamin A/C (LMNl). Furthermore provided is the use of a nucleic acid or antibody capable of detecting the expression level of BCLl lA .

These nucleic acids or antibodies can be used in the herein provided methods to determine whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, Antibodies to be used in this context are known in the art, like commercially available antibodies. Exemplary anti- LMNl antibodies to be used herein are poly-clonal rabbit antibodies HPA006660 (Atlas Antibodies, Stockholm, Sweden), and #2032 (Cell Signaling, Danvers, Massachusetts).

Preferably, the nucleic acid or oligonucleotide(s) is (are) about 15 to 100 nucleotides in length. A person skilled in the art is, based on his general knowledge and the teaching provided herein, easily in the position to identify and/or prepare (a) an oligo- or polynucleotide capable of detecting the expression level of Lamin A/C (LMNl) or the expression level of BCLl lA. In particular these oligo- or polynucleotides may be used as probe(s) in the detection methods described herein. A skilled person will know, for example, computer programs which may be useful for the identification of corresponding probes to be used herein. For example, the (Pre-)Lamin A C (LMNl) nucleic acid sequence (SEQ ID NO: 6) (or parts thereof) may be used in this context for identifying specific probes for detecting the expression level of Lamin A/C (LMNl). Exemplary nucleic acid sequences are available on corresponding databases, such as the NCBI database (www.ncbi.nlm.nih.gov/sites/entrez).

The present invention also relates to a kit useful for carrying out the herein provided methods, the kit comprising a nucleic acid or an antibody capable of detecting the phosphorylation status of one or more of B-cell lymphoma/leukemia 1 1A protein (BCLl 1 A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) and/or a nucleic acid or an antibody capable of detecting the expression of Lamin A/C (LMNl) and/or a nucleic acid or an antibody capable of detecting the expression of BCLl 1 A. The kit may comprise antibodies known in the art as described abve. Also envisaged herein is the use of the herein described kit for carrying out the herein provided methods.

The present invention provides a kit that may further comprise or be provided with (an) instruction manual(s). For example, said instruction manual(s) may guide the skilled person (how) to determine the (reference/control) phosphorylation status of one or more of B-cell lymphoma/leukemia 11 A protein (BCLl 1 A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) . Said instruction manual(s) may guide the skilled person (how) to determine the (reference/control) expression level of Lamin A/C (LMNl). Said instruction manual(s) may also guide the skilled person (how) to determine the (reference/control) expression level of BCLl 1A. Particularly, said instruction manual(s) may comprise guidance to use or apply the herein provided methods or uses.

The kit (to be prepared in context) of this invention may further comprise substances/chemicals and/or equipment suitable/required for carrying out the methods and uses of this invention. For example, such substances/chemicals and/or equipment are solvents, diluents and/or buffers for stabilizing and/or storing (a) compound(s) required for specifically determining the phosphorylation status of one or more of B-cell lymphoma/leukemia ΠΑ protein (BCLl lA), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) and/or the expression level of Lamin A/C (LMNl) and/or the expression level of BCLl 1 A.

The inhibitor may be administered as a single anti-tumor agent or in form of a combination therapy. The therapy used in said combination therapy may be chemotherapy or an anti-hormonal therapy. The chemotherapy may be anthracycline/taxane chemotherapy, therapy with an anti-metabolite agents, therapy with an anti-hormonal compound, therapy with an anti-estrogen, therapy with a tyrosine kinase inhibitor, therapy with a raf inhibitor, therapy with a ras inhibitor, therapy with a dual tyrosine kinase inhibitor, therapy with taxol, therapy with taxane, therapy with doxorubicin, therapy with adjuvant (anti-) hormone drugs, and/or therapy with cisplatin and the like. The inhibitor may be administered by any one of a parenteral route, oral route, intravenous route, subcutaneous route, intranasal route or transdermal route.

The present invention also relates to the use of an FLT3 inhibitor as defined herein for the preparation of a pharmaceutical composition for the treatment of a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient

(i) is not phosphorylated at one or more phosphorylation sites of one or more of B-cell lymphoma/leukemia 1 1 A protein (BCL1 1A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1 ), and/or Ran- binding protein 3 (RANBP3); and/or

The pharmaceutical composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, the method of administration, the scheduling of administration, and other factors known to practitioners. The "effective amount" of the pharmaceutical composition for purposes herein is thus determined by such considerations.

The skilled person knows that the effective amount of pharmaceutical composition administered to an individual will, inter alia, depend on the nature of the compound. For example, if said compound is a (polypeptide or protein the total pharmaceutically effective amount of pharmaceutical composition administered parenterally per dose will be in the range of about 1 μg protein /kg/day to 10 mg protein /kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg protein /kg/day, and most preferably for humans between about 0.01 and 1 mg protein /kg/day. If given continuously, the pharmaceutical composition is typically administered at a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1 -4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art.

Pharmaceutical compositions of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.

Pharmaceutical compositions of the invention preferably comprise a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous and intraarticular injection and infusion.

The pharmaceutical composition is also suitably administered by sustained release systems. Suitable examples of sustained-release compositions include semi -permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma- ethyl-L-glutamate (Sidman, U. et al, Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained release pharmaceutical composition also include liposomally entrapped compound. Liposomes containing the pharmaceutical composition are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal therapy.

For parenteral administration, the pharmaceutical composition is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

Generally, the formulations are prepared by contacting the components of the pharmaceutical composition uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The components of the pharmaceutical composition to be used for therapeutic administration should be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic components of the pharmaceutical composition generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The components of the phamiaceutical composition ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophiiized formulation for reconstitution. As an example of a lyophiiized formulation, 10-ml vials are filled with 5 ml of sterile- filtered 1% (w/v) aqueous solution, and the resulting mixture is lyophiiized. The infusion solution is prepared by reconstituting the lyophiiized compound(s) using bacteriostatic Water-for-Inj ection. The phosphorylation site can be one or more of the following phosphorylation sites:

a. SI 60 of EEPD1;

b. S630 of BCLl lA;

c. S333 of RANBP3;

d. S961 of RP3; and/or

e. S458 of LMNl .

The proliferative diseased cell can be determined to have the phosphorylation sites SI 60 of EEPD1; S630 of BCL1 1A; S333 of RANBP3; S458 of LMNl not phosphorylated; and the proliferative diseased cell can be determined to have the phosphorylation sites S961 of RP3 phosphorylated. Accordingly, the phosphorylation sites SI 60 of EEPD1 ; S630 of BCL11A; S333 of RANBP3; and S458 of LMNl can be not phosphorylated; and the phosphorylations sites S961 of RP3 can be phosphorylated.

The proliferative diseased cell can be determined to have Lamin A/C (LMNl) expressed at a decreased level in comparison to the control. The proliferative diseased cell can be characterized by Lamin A/C (LMNl) expression.

Herein provided is a method of treating a patient, said method comprising selecting a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is determined to have Lamin A/C (LMNl) expressed; and administering to the patient an effective amount of an FMS-like tyrosine kinase 3 (FLT3) inhibitor.

Further, herein provided is an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is characterized by Lamin A/C (LM l) expression.

The present invention relates to an FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is characterized by Lamin A C (LMNl) expression, wherein the treatment comprises determining the expression of Lamin A C (LMN 1 ) in a sample of a patient. The expression level of Lamin A/C (LMN1) can be decreased in comparison to the control. The expression level of Lamin A/C (LMN1) can be at least 2.5-fold, preferably at least 5-fold decreased in comparison to the control.

Exemplary FMS-like tyrosine kinase 3 (FLT3) inhibitors are quizartinib (AC220), midostaurin (PKC-412), sorafe ib (Bay-43-0006), 4SC-203, tandutimb (MLN-0518), sunitinib (SU-11248), and lestaurinib (CEP-701). Preferably, the FMS-like tyrosine kinase 3 (FLT3) inhibitor is quizartinib (AC220). The FLT3 inhibitor can be a selective FMS-like tyrosine kinase 3 (FLT3) inhibitor.

As mentioned, the patient can be suspected to suffer from neoplasia, is suffering from neoplasia or being prone to suffer from neoplasia. The neoplasia can be a malignant neoplasia. The malignant neoplasia can be leukemia.

Leukemia is considered in the art as a cancer that starts in blood-forming tissue such as the bone marrow and causes large numbers of blood cells to be produced and enter the bloodstream. The leukemia cells can build up in the blood and bone marrow so there is less room for healthy white blood cells, red blood cells, and platelets. When this happens, infection, anemia, or easy bleeding may occur. The leukemia cells can spread outside the blood to other parts of the body, including the central nervous system (brain and spinal cord), skin, and gums. Sometimes leukemia cells form a solid tumor called a granulocytic sarcoma or chloroma.

The leukemia can be myeloid leukemia, such as acute myeloid leukemia (AML).

Adult acute myeloid leukemia (AML) is a cancer of the blood and bone marrow. This type of cancer usually gets worse quickly if it is not treated. It is the most common type of acute leukemia in adults. AML is also called acute myelogenous leukemia, acute myeloblastic leukemia, acute granulocytic leukemia, or acute nonlymphocytic leukemia.

Childhood acute myeloid leukemia (AML) is a cancer of the blood and bone marrow. Cancers that are acute usually get worse quickly if they are not treated. Cancers that are chronic usually get worse slowly. Acute myeloid leukemia (AML) is also called acute myelogenous leukemia, acute myeloblastic leukemia, acute granulocytic leukemia, or acute nonlymphocytic leukemia. In AML, the myeloid stem cells usually become a type of immature white blood cell called myeloblasts (or myeloid blasts). The myeloblasts in AML are abnormal and do not become healthy white blood cells. Sometimes in AML, too many stem cells become abnormal red blood cells or platelets. These abnormal white blood cells, red blood cells, or platelets are also called leukemia cells or blasts. Leukemia cells can build up in the bone marrow and blood so there is less room for healthy white blood cells, red blood cells, and platelets. When this happens, infection, anemia, or easy bleeding may occur. The leukemia cells can spread outside the blood to other parts of the body, including the central nervous system (brain and spinal cord), skin, and gums.

There are subtypes of AML based on the type of blood cell that is affected. The treatment of AML is different when it is a subtype called acute promyelocyte leukemia (APL) or when the child has Down syndrome. Most AML subtypes are based on how mature (developed) the cancer cells are at the time of diagnosis and how different they are from normal cells.

Acute promyelocyte leukemia (APL) is a subtype of AML that occurs when parts of two genes stick together. APL usually occurs in middle-aged adults. Symptoms of APL may include both bleeding and forming blood clots.

The leukemia can be lymphoid leukemia, such as acute lymphoid leukemia (ALL).

Childhood acute lymphoblastic leukemia (also called acute lymphocytic leukemia or ALL) is a cancer of the blood and bone marrow. This type of cancer usually gets worse quickly if it is not treated. It is the most common type of cancer in children.

Adult acute lymphoblastic leukemia (ALL; also called acute lymphocytic leukemia) is a cancer of the blood and bone marrow. This type of cancer usually gets worse quickly if it is not treated.

In acute lymphoid leukemia (ALL), too many stem cells become lymphoblasts, B lymphocytes, or T lymphocytes. These cells are also called leukemia cells. These leukemia cells do not work like normal lymphocytes and are not able to fight infection very well. Also, as the number of leukemia cells increases in the blood and bone marrow, there is less room for healthy white blood cells, red blood cells, and platelets. The neoplasia may be a myelodysplastic syndrome, such as refractory anemia with excess of blasts (RAEB I or RAEB II).

The present invention relates to a method for testing a proliferative diseased cell of a neoplasia patient to determine whether said proliferative diseased cell is responsive to FMS-like tyrosine kinase 3 (FLT3) inhibitor therapy, the method comprising testing a sample of a patient for whom FMS-like tyrosine kinase 3 (FLT3) inhibitor therapy is contemplated to determine the phosphorylation status therein of one or more of B-cell lymphoma/1 eukemia 1 1A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exomiclease/phosphatase family domain- containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) wherein said phosphorylation status is indicative of responsiveness to FMS-like tyrosine kinase 3 (FLT3) inhibitor therapy.

The present invention relates to a method for testing a proliferative diseased cell of a neoplasia patient to determine whether said proliferative diseased cell is responsive to FMS-like tyrosine kinase 3 (FLT3) inhibitor therapy, the method comprising obtaining a sample of a patient for whom FMS-like tyrosine kinase 3 (FLT3) inhibitor therapy is contemplated, and testing the sample to determine the phosphorylation status therein of one or more of B-cell lymphoma/1 eukemia 11A protein (BCL1 1A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) wherein said phosphorylation status is indicative of responsiveness to FMS-like tyrosine kinase 3 (FLT3) inhibitor therapy.

As used herein, the terms "comprising" and "including" or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms "consisting of and "consisting essentially of."

Thus, the terms "comprising"/"including"/"having" mean that any further component (or likewise features, integers, steps and the like) can be present.

The term "consisting of means that no further component (or likewise features, integers, steps and the like) can be present. The term "consisting essentially of or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method.

Thus, the term "consisting essentially of means that specific further components (or likewise features, integers, steps and the like) can be present, namely those not materially affecting the essential characteristics of the composition, device or method. In other words, the term "consisting essentially of (which can be interchangeably used herein with the term "comprising substantially"), allows the presence of other components in the composition, device or method in addition to the mandatory components (or likewise features, integers, steps and the like), provided that the essential characteristics of the device or method are not materially affected by the presence of other components.

The term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts.

As used herein the term "about" refers to ± 10%.

The following abbreviations are used herein:

AUROC: Area Under the Receiver Operating characteristic Curve; CV: Cross Validation; FDR: false discovery rate; FFPE: formalin-fixed and paraffin-embedded; LOOCV: Leave-One-Out Cross Validation; NSCLC: Non-Small Cell Lung Cancer; SILAC: Stable Isotope Labeling by Amino acid in cell Culture; SVM: Support Vector Machine. The abbreviated terms and the respective full terms can be used interchangeably herein. The present invention relates to the following items:

1. A method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of one or more of B-cell lymphoma leukemia 11A protein (BCLl lA), Lamin A C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor. . The method of item 1 , wherein said phosphorylation status is the presence or absence of phosporylation at one or more phosphorylation sites of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), B-cell lymphoma/leukemia 11 A protein (BCLl lA), Ran-binding protein 3 (RANBP3), GTPase regulator (RP3) and/or Lamin A/C (LMN1).

3. The method of item 2, wherein the absence of phosporylation at one or more phosphorylation sites of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), B-cell lymphoma/leukemia 11A protein (BCLl lA), Ran-binding protein 3 (RANBP3), and/or Lamin A/C (LMN1) is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor. . The method of item 2 or 3, wherein the presence of phosporylation at one or more phosphorylation sites of GTPase regulator (RP3) is indicative of responsiveness to said FMS- like tyrosine kinase 3 (FLT3) inhibitor. . The method of any one of items 2 to 4, wherein said phosphorylation site is one or more of the following phosphorylation sites:

a. SI 60 of EEPDl;

b. S630 of BCLl lA;

c. S333 of RANBP3;

d. S961 of RP3; and/or

e. S458 of LMNl . The method of any one of items 1 to 5, further comprising determining the expression level of Lamin A/C (LMNl). The method of item 6, wherein a decrease in said expression level in comparison to the control is indicative of the responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor. The method of any one of items 1 to 7, wherein said FMS-like tyrosine kinase 3 (FLT3) inhibitor is selected from the group consisting of quizartinib (AC220), midostaurin (PKC- 412), sorafenib (Bay-43-0006), 4SC-203, tandutinib (MLN-0518), sunitinib (SU-11248), and lestaurinib (CEP-701). The method of any one of items 1 to 8, wherein the patient is suspected to suffer from myeloid leukemia, suffering from myeloid leukemia or being prone to suffer from myeloid leukemia, in particular acute myeloid leukemia (AML). An FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient

(i) is not phosphorylated at one or more phosphorylation sites of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), B-cell lymphoma/leukemia 11A protein (BCLl lA), an-binding protein 3 (RANBP3), and/or Lamin A C (LM l); and/or

(ii) is phosphorylated at one or more phosphorylation sites of GTPase regulator (RP3), optionally wherein the treatment comprises determining the phosphorylation status at a phosphorylation site of one or more of B-cell lymphoma/leukemia 1 1A protein (BCLl lA), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3) and/or GTPase regulator (RP3) in a sample of a patient. The FMS-like tyrosine kinase 3 (FLT3) inhibitor of item 10, wherein said phosphorylation site is one or more of the following phosphorylation sites:

a. S160 ofEEPDl ; b. S630 ofBCLl lA;

c. S333 of RANBP3;

d. S961 of RP3; and/or

e. S458 of LMNl .

12. The FMS-like tyrosine kinase 3 (FLT3) inhibitor of item 10 or 1 1, wherein the phosphorylation sites S160 of EEPD1 ; S630 of BCL1 1A; S333 of RANBP3; and S458 of LMN1 are not phosphorylated; and wherein the phosphorylation site S961 of RP3 is phosphorylated.

13. The FMS-like tyrosine kinase 3 (FLT3) inhibitor of any one of items 10 to 12, wherein said proliferative diseased cell is characterized by Lamin A/C (LMN1) expression.

14. The FMS-like tyrosine kinase 3 (FLT3) inhibitor of any one of items 10 to 13, wherein said FMS-like tyrosine kinase 3 (FLT3) inhibitor is selected from the group consisting of quizailinib (AC220), midostaurin (PKC-412), sorafenib (Bay-43-0006), 4SC-203, tandutinib (MLN-0 18), sunitinib (SU- 11248), and lestaurinib (CEP-701).

15. The FMS-Hke tyrosine kinase 3 (FLT3) inhibitor of any one of items 10 to 14, wherein the neoplasia patient is suspected to suffer from myeloid leukemia, is suffering from myeloid leukemia or is being prone to suffer from myeloid leukemia, in particular acute myeloid leukemia (AML).

It is envisaged that any of the above items (and combinations thereof) can be combined with any of the items/aspects provided herein in accordance with the present invention.

The present invention is further described by reference to the following non-limiting figures and examples. Unless otherwise indicated, established methods of recombinant gene technology were used as described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001) ) which is incorporated herein by reference in its entirety. The Figures show:

Figure 1. Workflow of processing bone marrow aspirates and global quantitative phosphoproteome analysis.

The leukemia cells were isolated using density-gradient centrifugation and stored as vital cells for further processing at -80°C. Equal amounts of lysates from blasts and Super- SILAC-standard were mixed. Proteins were extracted and digested with trypsin. The resulting peptides were separated into twelve fractions by SCX chromatography and the phosphopeptides were enriched using MAC. Finally, high resolution LC-MS/MS data were processed using the MaxQuant software.

Figure 2. Comparison of responder and non-responder classes.

A: Scatter plot shows the mean log-ratios (AML sample vs. spike-in SILAC reference) for the responder (y-axis) and non-responder (x-axis) samples. Each dot represents one phosphorylation site. The three significantly differential sites are marked by circles. B: Log-ratios for Y694 (STAT5A) or Y699 (STAT5B) across all training samples.

Figure 3. Identification of predictive phospho-signature.

A: Final phospho-signature consisting of 5 phosphosites shown in Example 1. Each pair of boxes corresponds to one phosphosite. The left (right) box represents the responder (non-responder) samples. On each box, the central mark is the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points not considered outliers, and outliers are marked individually with crosses. B: Cross-validation results represented by probability for assignment to the responder class. Responders (left half) are predicted correctly if they get assigned a probability >0.5; non-responder (right half) are correct if they are assigned a probability <0.5. C: Phosphorylation degree of the final 5 selected marker sites (columns) across the 2 training samples (rows). Rows are the 12 training sample, columns are the phosphosites ordered by their importance ranks (left is the best). A corresponding description is found in Figure 3C. Missing values are shown in white. D: Similar as C, but degree of phosphorylation is depicted with triangles. Orientation of triangles show the direction of regulation compared to the Super-SILAC reference: up (down) corresponds to ratios greather (smaller) than 1. The size of the triangles corresponds to the absolute value of the log ratio vs. the Super-SILAC reference. Figure 4. Validation of the phosphosignature.

Validation results represented by probability for assignment to the responder class. Responders (left half) are predicted correctly if they get assigned a probability >0.5; non-responder (right half) are correct if they are assigned a probability <0.5.

Figure 5. Correlation between phosphorylation and protein expression.

A: EEPD1 (S160) across six validation samples. B: LMN1 (S458) across all samples.

Figure 6, Prediction obtained if single phosphorylation site instead of combined signature is used.

Prediction result is represented by probability for assignment to the responder class. Responders (left half) are predicted correctly if they get assigned a probability >0.5; non-responder (right half) are correct if they are assigned a probability <0.5.

Figure 7. Normalized ratio of phosphorylation between peripheral blood and bone marrow sample of patients AML014, AML020, and AML025.

Each data point represents one phosphorylation site and one sample.

Figure 8. Correlation of phosphorylation of two different sites on the same protein.

Each plot represents one particular phosphorylation site. The x-axis shows the super-SILAC ratio for the site that was included in the phospho- signature; the y-axis shows the ratio for a different site on the same protein. Each dot corresponds to one particular AML sample, r is the Pearson correlation coefficient, p the corresponding p-value.

Figure 9: Phosphorylation of BAD.

Log ratios of phosphorylation of site SI 18 of BAD in investigated AML samples vs. Super-SILAC reference.

The Examples illustrate the invention. Example 1: Phosphorylation Markers predict treatment efficacy of neoplasia with a FMS- like tyrosine kinase 3 inhibitor

Material and Methods

Specimen collection and processing

Bone marrow aspirates of 22 patients suffering from AML were collected. 21 patients were enrolled in the phase II clinical trial of AC220 monotherapy in AML with FLT3-ITD mutations at the Goethe University (Frankfurt, Germany), the Medizinische Hochschule (Hannover, Germany), the Johns Hopkins University (Baltimore, Maryland), and the University of Pennsylvania (Philadelphia, Pennsylvania). The 22^nd patient was FLT-ITD negative and was therefore not enrolled in the trial. Details on the clinical trial (ACE) are reported elsewhere (Cortes, Perl et al. 2011). Samples were collected pre-treatment. All patients gave informed consent according to the Declaration of Helsinki to participate both in the clinical trials and the collection of samples. Use of bone marrow aspirates was approved by the respective local ethical committee at each individual institution.

The patients were divided into two collections. The first collection of in total 13 patients consists of samples from Goethe University, from Medizinische Hochschule, and a first set of 5 samples from University of Pennsylvania. These samples were used for training. The collection also contains the patient who was not enrolled and for whom the AC220-response is thus unknown. The second collection of in total 9 patients consists of samples from Johns Hopkins University and a second set of 6 samples from University of Pennsylvania. These samples were used for validation. Both collections were processed in separate batches in the following. All clinics followed a standard operating procedure for preparation of the bone marrow aspirates that was defined based on the results of a previous study. In brief, a maximum of 8 ml bone marrow aspirate was collected in 2 ml of ethylenediaminetetraacetic acid (EDTA) or 10% heparin, processed by FICOLL separation and then stored in 10%DMSO/10% FCS in liquid nitrogen.

Patients with complete remission (CR), complete remission with incomplete haematological recovery (CRi), complete remission with incomplete platelate recovery (CRp), and partial remission (PR) were counted as responder. Patients with stable disease (SD) or no response were counted as non-responder. Spike-in reference

The human AML cell lines OCI-M1, NB4, and MV4-1 1 were chosen as Super-SILAC reference (Geiger, Cox et al. 2010). OCI-M1 and NB-4 were obtained from Christian JunghanB' group (University Rostock, Germany). MV4-11 was obtained from the DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). NB-4 and MV4-1 1 were cultivated in RPMI, 10% foetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate and penicillin/streptomycin (PAA, Colbe, Germany). OCI-M1 was cultivated in IMDM, 10% foetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin (PAA, Colbe, Germany), Metabolic labelling of the cell lines was performed using SILAC (stable isotope labelling by amino acids in cell culture (Ong, Blagoev et al. 2002)). Cells were cultivated in media containing SILAC-RPMI or IMDM (PAA) and dialysed FBS (PAA). L-lysine and L-arginine were replaced by heavy isotope- labelled L-¹³C₆ ¹⁵N₂-lysine (Lys-8) and L-¹³C₆ ¹⁵N₄-arginine (Arg-10). Isotope- labelled amino acids were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Cells were cultivated for a minimum of six doubling times to obtain an incorporation efficiency for the labelled amino acids of at least 95%. The labelled cells were lysed, aliquoted, and stored at -80°C.

Phosphoproteomics workflow

Viable stocks of frozen AML cells were thawed on ice, centrifuged (3 min, 2,000 rpm, 4°C) and then lysed in ice-cold lysis buffer (8 M urea, 50 mM Tris pH 8.2, 10 mM sodium pyrophosphate, 5 mM EDTA, 5 mM EGTA, 10 mM sodium fluoride, 10 mM β-glycerophosphat, 2 mM sodium orthovanadate, phosphatase inhibitor cocktail 2 and 3 (Sigma, 1 :100 (v/v)) and Complete Protease Inhibitor Cocktail Tablets (Roche). After sonication the cell debris was removed by centrifugation (10 min at 13,000 rpm, 4°C) and the protein concentration was determined utilizing the Bradford Protein Assay (BIO-RAD). Equal protein amounts of the Super-SILAC reference were added and subsequently subjected to reduction (10 mM dithiothreitol, 30 min 37°C) and alkylation (50 mM iodoacetamide, 30 min RT). The alkylation reaction was quenched by adding 20 mM DTT. Proteins were initially digested with lysyl enpopeptidase (Wako, 1 :200 (w/w)) for 4 hours then diluted 5- times with 20 mM Tris pH 8.2 prior to overnight proteolytic cleavage with trypsin (Promega, 1 :100 (w/w)). The peptide mixtures were acidified by addition of TFA to a final concentration of 0.5 % and subsequently desalted via CI 8 Sep-Pak columns (Waters). Peptides were eluted with 50% acetonitrile, 0.5% acetic acid, snap frozen in liquid nitrogen and lyophilized. Phosphorylated and non-phosphorylated peptides were initially fractionated by strong-cation- exchange (SCX) chromatography based on a previously described protocol (Villen and Gygi 2008) using a PolySULFOETHYL A column (200x2.1 mm, 200 A pore size and 5 mm particle size; PolyLC) operated with an Akta Purifier system (GE Healthcare). Briefly, the dried peptides were reconstituted in 100 μΐ SCX buffer A (5 mM K HP0₄, pH 2.7, 30% acetonitrile) and loaded onto the SCX column. The peptides were separated by a linear gradient from 0 to 25% SCX buffer B (buffer A supplemented with 500 mM KCl) over 32 min at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected across the gradient and combined to 12 distinct samples. These samples were then lyophilized and the dried peptides were subsequently reconstituted in 1 ml of 0.1% TFA and desalted using CI 8 reversed phase cartridges (Waters) as described by the manufacture. The desalted peptides were eluted with 50% acetonitrile, 0.5% acetic acid and lyophilized again.

Dried phosphopeptides of each fraction were reconstituted in IMAC binding buffer (40% acetonitrile, 25 mM formic acid) and phosphopeptides were captured using PHOS-Select® iron affinity beads (Sigma) based on the protocol by Villen et.al. (Villen and Gygi 2008). Briefly, 5 μΐ of equilibrated IMAC beads were loaded onto in-house made IMAC-C18-STAGE-Tips (IMAC- StageTips) and the peptide samples were loaded by centrifugation (3,000 rpm). After washing with 1 % formic acid, phosphopeptides were eluted onto the CI 8 frit with 500 mM K₂HP0₄. Phosphopeptides were then eluted with 50% acetonitrile, 0.5% acetic acid after additional washing steps with 0.1 % TFA and 0.5 % acetic acid and dried in a vacuum concentrator (Eppendorf). For MS-anaylsis phosphopeptides were reconstituted in 0.5% acetic acid.

LC-MS/MS Analysis

All LC-MS/MS analyses were performed on an LTQ-Orbitrap Velos (Thermo Fisher Scientific). The samples were loaded by an Proxeon easy nano LC II system (Thermo Fisher Scientific) on a 15 cm fused silica emitter (New Objective) packed in-house with reversed phase material (Reprusil- Pur C18-AQ, 3 μιη, Dr. Maisch GmbH) at a maximum pressure of 275 bar. The bound peptides were eluted by a gradient from 10% to 60% of solvent B (80% acetonitrile, 0.5%» acetic acid) at a flow rate of 200 nl/min and sprayed directly into the mass spectrometer by applying a spray voltage of 2.2 kV using a nanoelectrospray ion source (ProxeonBiosystems). The mass spectrometer was operated in the data dependent mode to automatically switch between MS and MS/MS acquisition. To improve mass accuracy in the MS mode, the lock-mass option was enabled as described (Olsen, de Godoy et al. 2005). Full scans were acquired in the orbitrap at a resolution R = 60,000 and a target value of 1,000,000 ions. The fifteen most intense ions detected in the MS scan were selected for collision induced dissociation in the LTQ at a target value of 5000 ion counts. The resulting fragmentation spectra were also recorded in the linear ion trap. To improve complete dissociation of phosphopeptides, the multi-stage activation option was enabled for all MS-analyses of phosphopeptide-enriched samples by applying additional dissociation energy on potential neutral loss fragments (precursor ion minus 98, 49 and 32.7 m/z) (Schroeder, Shabanowitz et al. 2004). Ions that were once selected for data dependent acquisition were dynamically excluded for 90 sec for further fragmentation. General used mass spectrometric settings were: spray voltage, 2.2 kV; no sheath and auxiliary gas flow; heated capillary temperature, 230°C; normalized collision energy, 35% and an activation q = 0.25.

MaxQuant analysis

MS raw files from the training and the validation collection were processed separately with MaxQuant (version 1.2.2.2) (Cox and Mann 2008) applying the Andromeda search engine (Cox, Neuhauser et al. 201 1). The human U IPROT database (version: 08.2011) was used comprising 125,676 database entries including the UNIPROT splice variants database. The minimal peptide length was set to 6 amino acids, trypsin was selected as proteolytic enzyme and maximally 2 missed cleavage sites were allowed. Carbamidomethylation of cysteine residues was set as fixed modification while oxidation of methionine, protein jV-acetylation as well as phosphorylation of serine, threonine and tyrosine residues was allowed as variable modifications. As MaxQuant automatically extracts isotopic SILAC peptide doublets, the corresponding isotopic forms of lysine and arginine were automatically selected. The maximal mass deviation of precursor and fragment masses was set to 20 ppm and 0.5 Da before internal mass recalibration by MaxQuant. A false discovery rate (FDR) of 0.01 was selected for proteins, peptides, and phosphorylation sites. The MaxQuant results were uploaded to the MaxQB database (version 2.9) (Schaab, Geiger et al. 2012) for further analysis.

Data pre-processing

The regulation of a phosphosite is provided as ratio of the site's abundance between the spike-in SILAC reference (heavy) and the marrow samples (light). The normalized ratios provided by MaxQuant were log-transformed (base 10) for further analysis. Sites that satisfy the constraints Localization Probability^ 0.75 and Score Diff>=5 were considered to be sufficiently reliable (class I sites (Olsen, Blagoev et al. 2006)). Furthermore, sites that are flagged as Reverse or Contaminant hits were excluded.

Mean-Rank test

Suppose a matrix M with R columns (replicates) and N rows (features, e.g. genes, proteins, phosphorylation sites). Let qi(f) be the log-ratio for feature f (with f = 1, N) in replicate i (with i = 1, R). Based on this matrix M, the rank ri(f) of each feature can be determined by sorting the log-ratios in each replicate. Subsequently, the mean rank is calculated for each feature across all replicates. Similar to the approach of Zhou (Zhou, Cras-Meneur et al. 2007) the mean rank statistic is motivated by the random ordering theorem, i.e. under the null hypothesis HO that no features are either up- or down-regulated, no features can rank consistently high or low across all replicates, and therefore no extreme (very small or very high) mean rank values can be expected. On the contrary, while Zhou et al. require features to rank top or bottom consistently across all replicates, the mean rank statistic may tolerate some moderate outliers.

Identification and validation of phospho-signature

The statistical analysis of the data was performed in Matlab (The Mathworks, Natick, MA). The Mean-Rank test was applied to find differentially abundant phosphorylation sites between two groups of samples. The Mean-Rank test is more powerful than tests based on the parametric or non- parametric t-test or Wilcoxon rank-sum test, when only few replicates are available. It controls for the false-discovery-rate (FDR) without requiring additional correction for multiple hypothesis testing. For this analysis only phosphosites with values in at least two thirds of the experiments in each group were considered. The FDR was set to 0.10.

The details of the workflow for identification of a predictive phospho-signature was described elsewhere (Klammer, aminski et al. 2012). In brief, the Mean-Rank test was used in combination with the ensemble feature selection method (Abeel, Helleputte et al 2010). The number of features was fixed to five. The selected features were used to train a SVM with linear kernel and the parameter C=l . Missing data in the training samples were imputed by the mean of the respective class. The prediction accuracy and the area under the receiver operating characteristic curve (AUROC) were determined on the training set by leave-one-out cross-validation. Missing data in the test sample were imputed by the mean of the two class means. Prediction probabilities were calculated from a sigmoid model that was fitted to the SVM output of the respective training data (Lin, Lin et al. 2007). In the sigmoid function p. = , l + exp( ;. + B) where p_t is the probability of the sample being a non-responder and fi the SVM output, only parameter A was optimized; parameter B was fixed to 0, so that points on the separating hyper plane are assigned a probability of 0.5.

The complete training data set was used to select five predictive features and to train the final SVM, which was then applied to the classification of new samples from the validation set. Missing data were again imputed by the mean of the two class means.

Sample preparation for proteome analysis

For global proteome analysis of selected samples the twelve flow-through fractions of the IMAC- hased phosphopeptide enrichments were collected, experiment-wise combined, lyophilized and desalted using CI 8 reversed phase cartridges (Waters). High pH reversed phase separation of peptides was performed based on a previously described protocol (Wang, Yang et al. 2011). Briefly, peptides were reconstituted in buffer A (20 mM ammonium formate, ph 10) and loaded to an XBridge CI 8, 250 ^χ 4.6 mm analytical column (Waters) operated with the Akta Purifier system (GE Healthcare). Buffer B was buffer A supplemented with 80% acetonitrile. By applying a segmented gradient from 7.5 to 30 % buffer B for 15 min and 30 to 55 % buffer B for 5 min followed by 5 min 100% buffer B peptides were separated at a flow rate of 1 ml/min. Fractions of 1 ml were collected throughout the gradient and combined in a non-linear way to result in twelve samples for each individual experiment. Samples were then frozen in liquid nitrogen and lyophilized. For MS-analysis the peptides were reconstituted in 0.5 % acetic acid.

Results

We aimed to identify phosphorylation events that predict clinical response with high accuracy, especially in the group of FLT3-ITD-positive patients. To this end, a global, quantitative phosphoproteome analysis of AML cells derived from patient bone marrow was established. Samples from 12 patients before treatment with AC220 were analyzed and the obtained phosphoproteomics data was employed to discover a predictive signature of five phosphorylation markers. The predictive power of this signature was evaluated by cross-validation and by testing it on an independent set of 9 additional patient samples.

Bone-marrow samples of twenty-two patients with AML were collected.

Bone marrow aspirates of twenty-two AML patients were collected before treatment with AC220 (Tab. 1). Twenty-one patients were enrolled in the ACE trial at the Goethe University (Frankfurt, Germany), the Medizinische Hochschule (Hannover, Germany), the Johns Hopkins University (Baltimore, Maryland), and the University of Pennsylvania (Philadelphia, Pennsylvania). One additional patient (Goethe University) was diagnosed as FLT3-ITD negative and was therefore not enrolled in the trial. We processed the aspirates according to the sample preparation workflow that was established previously (Fig. 1). All samples contained between 6 and 50 million blasts. In average 316 μ protein could be extracted.

The twenty-one samples from patients treated with AC220 were divided into two collections. The first collection (12) was processed at the beginning of the study and was used for identification of the predictive phosphorylation-signature. The second collection (9) was processed towards end of the study and was used for validating the signature. All patients with complete response (including CRi and CRp) or partial response were counted as responder (6/12 in the training collection and 6/9 in the validation collection).

Phosphoproteomics profiling reveals differences between the two patient classes.

The blasts from the 22 subjects in the training collection were analysed with respect to their global phosphoproteomes. Generally, SILAC allows accurate and robust quantification in mass- spectrometry based proteomics experiments (Ong, Blagoev et al. 2002). However, it requires complete metabolic labelling of the proteome and is therefore restricted to cultured cells and a few model organisms. To enable quantitative comparisons between clinical samples, the so-called Super-SILAC approach was applied (Geiger, Cox et al. 2010), which was recently extended to phosphoproteomic analysis (Monetti, Nagaraj et al. 2011; Klammer, Kaminski et al. 2012). Three AML cell lines OCI-M1 , NB4, and MV4-11 were selected that have different FAB (French- American-British) types and should cover a large part of phosphorylation events observable in clinical AML samples. The cell lines were grown in heavy SILAC medium (Arg¹⁰/Lys⁸), lysed and mixed to generate the Super-SILAC standard. Equal protein amounts of the blasts and the standard were mixed and subjected to a global, quantitative phosphoproteomics workflow using strong cation exchange chromatography (SCX) and immobilised metal ion affinity chromatography (IMAC). Finally the enriched samples were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS, see Experimental Procedures for more details, Fig. 1).

In total, 13,236 phosphosites were identified in the training collection. Of these 7,831 were rated as class-I sites, i.e. sites that could be identified and localized with high confidence (Olsen, Blagoev et al. 2006). Only these sites were used in the following analyses. The frequency distribution of phosphorylated residues (serine: 90.1 %, threonine 9.4%, tyrosine: 0.5%) is similar to the frequency distributions observed in other studies (Olsen, Blagoev et al. 2006; Klammer, Kaminski et al. 2012). 98% of all identified class-I sites could also be quantified in at least one sample. Moreover, 68% of all AML to Super-SILAC ratios are in the range between 0.25 and 4. Since the estimation of larger ratios from SILAC MS raw data is more inaccurate than of smaller ones, this is of pivotal importance.

It was first investigated whether differentially regulated phosphosites can be identified when comparing responder and non-responder samples (Fig. 2A). Only class-I sites with data values in at least two thirds of the experiments were used (2,1 39 sites with approximately 10.6% missing values on average). Indeed, application of the mean-rank test revealed three significantly regulated sites at an FDR of 10% (see Tab. 2). The first regulated site (SI 60) is located on the endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1). The protein carrying the second phosphorylation site (S630), B-cell lymphoma leukemia 1 1 A (BCL1 1A), functions as a myeloid and B-cell proto-oncogene and may play a role in leukemogenesis and hematopoiesis (UniProtKB) (UniProt Consortium 2011). Furthermore, the expression of BCL11A is associated with poor outcome of AML patients (Yin, Delwel et al. 2009). The third phosphorylation site (S333) is located on Ran-binding protein 3 (RANBP3). RANBP3 mediates nuclear export of Smad2/3 and thereby inhibits TGF-beta signalling (Dai, Lin et al. 2009). Furthermore, the Ras/ERK/RSK and the PI3K/AKT signalling pathways regulate activity of RANBP3 (Yoon, Shin et al. 2008). Both pathways are activated in FLT3-ITD positive cells (Choudhary, Olsen et al. 2009). All three regulated phosphorylation sites were detected in previous mass- spectrometry based phosphoproteomics studies (PhosphoSitePlus) (Hornbeck, Chabra et al. 2004). However no function has been described for these phosphorylations so far. Next phosphorylation sites known to be involved in FLT3-ITD induced signalling were investigated. None of the auto-phosphorylated tyrosine-sites of FLT3 were detected in this study. Two serine sites (S762 and S979) could be quantified in a few samples only. However, phosphorylations of the protein kinases STAT5A and STAT5B, which are known to be activated by FLT3-ITD (Hayakawa, Towatari et al. 2000; Mizuki, Fenski et al. 2000; Choudhary, Olsen et al. 2009), could be identified and quantified. The tyrosine sites Y694 (STAT5A) and Y699 (STAT5B) show no differential phosphorylation between the responder and the non-responder classes (Fig. 2B).

A predictive phospho-signature was identified from the training samples.

As a next step a combination of phosphorylation sites was identified that allows the prediction of responsiveness using a supervised machine learning approach. The workflow for detecting phospho-signatures developed by Klammer et al. was applied (Klammer, Kaminski et al. 2012). Only phosphorylation sites that were quantified in at least two thirds of the samples in each class were used as potential features. For the training of the final predictor, 2,119 phosphorylation sites were kept after removing the sites with too many missing data. Further selection of predictive features is important because of two reasons: firstly, the reduction of dimensionality avoids over- fitting of the resulting predictor. Secondly, and maybe even more important, a smaller set of features can be easier applied in the clinic. Because of the small number of samples in the training data set, we omit the additional cross-validation loop for optimizing the number of features here. Rather, we fixed the number to five features, which is a reasonable signature size to be measured with tests applicable in the clinic, such as multiple-reaction-monitoring (MRM), Elisa-based assays, or immunohistochemistry. The predictive features were selected using the Mean-Rank test combined with the ensemble method (Abeel, Helleputte et al 2010). Standard feature selection methods tend to select very different sets of features depending on the samples that are used for training (Ein-Dor, Kela et al. 2005). The ensemble method tries to solve this issue by selecting features that repeatedly rank high when bootstrapping the training samples multiple times. The selected phosphorylation sites were used to train a support-vector machine (SVM) with linear kernel. SVMs have been shown to perform favourable compared to other methods in a number of studies and they have been successfully applied to similar data several times (e.g. see Ramaswamy, Tamayo et al. 2001; Hutter, Schaab et al. 2004; Thuerigen, Schneeweiss et al. 2006). The final phospho-signature of five phosphorylation sites (Tab. 3) strongly separates the classes of responder and non-responder samples (Fig. 3A and 3C). Three of the five phosphorylation sites (EEPD1-S160, BCL11A-S630, RANBP3-S333) were already identified as significantly regulated between responder and non-responder samples by using the same significance test, supplemented by the ensemble method, for ranking the features.The fourth phosphorylation site (S961) is located on the x-linked retinitis pigmentosa GTPase regulator (RP3). RP3 is predicted to be a guanine- nucleotide releasing factor and plays a role in ciliogenesis (UniProtKB). In contrast to the other four markers, RP3 is stronger phosphorylated in the responder group than in the non-responder group. Lamins A/C (LMN1) form the nuclear lamina and play an important role in regulation of nuclear structure during cell cycle and of gene transcription (Meier, Muller et al. 1997). The expression of LMN1 has recently been suggested as marker for increased risk of death from colorectal cancer (Willis, Cox et al. 2008). Here, strong phosphorylation of serine S458 correlates with insensitivity to treatment of AML with AC220. Again, all five signature sites were detected in previous mass- spectrometry based phosphoproteomics studies (PhosphoSitePIus) (Hornbeck, Chabra et al. 2004), but no function has been described for these sites so far. All five sites were identified and localized with high confidence (p>0.98).

The prediction performance of the phospho-signature was determined by leave-one-out cross validation (LOOCV). Previous studies showed that CV, including LOOCV, estimates the true prediction accurately and with low bias (Molinaro, Simon et al. 2005). In each iteration of cross- validation the feature selection and the training of the SVM is repeated on the training set reduced by the respective test sample. Only one sample (AML008) was misclassified as responder (Fig. 3B). The corresponding prediction accuracy is 92%. The area under the receiver operating characteristic curve (AUROC) is 88%. The only misclassified sample is AML008. In agreement with the prediction of the phospho-signature, the patient showed a marked reduction in marrow blasts (from 95% to 5-10%). Nevertheless he always had circulating blasts (5-10%) and was therefore not counted as responder. Each dot in Figure 2B represents the averaged prediction probability for this sample when all other samples were used for feature selection and SVM training. The larger the distance to the cut-off probability of 0.5, the more confident the prediction is.

The final signature was applied to the sample AMLOOl, for which the response was unknown. AMLOOl is FLT3-ITD negative and was therefore not enrolled in the trial. He was predicted to be a responder (p=0.66). This shows that AML001 could be a responder even though AML001 is wild- type FLT3.

As a further validation of our classification workflow, we wanted to prove that the data is not over- fitted, i.e. that we can expect a similar classi ication performance on validation samples not used for training. To this end, we applied the cross-validation workflow to randomized class labels. Strikingly, the prediction accuracy was only 50% (AUROC=0.53), which is almost exactly what one would expect if predicting the classes by chance.

The phospho-signature was validated in independent samples.

Finally the identified phospho-signature was applied to predict responsiveness of the nine additional validation samples. These samples were obtained from patients from the John-Hopkins University (3) and the Pennsylvania University (6). All nine samples were processed separately from the samples in the training collection. In particular, the MaxQuant processing was performed separately for the training and the validation collections. Seven out of the nine samples were predicted correctly (Fig. 4), one responder (AML031) and one non-responder (AML033) were misclassified. AML033 is indeed a borderline candidate. The patient's FLT3-ITD positive cells were sensitive and cleared by the drug treatment. However the patient progressed with a FLT3 wild- type clone. Because of this ambiguous call, we decided to not count this sample. The resulting sensitivity on the validation samples is 83% and the specificity 100% (67% with AML033). The corresponding accuracy is 88% (78% with AML033) and therefore comparable to the accuracy determined in cross-validation. This substantiates the surprising quality of the herein provided phosphosignature, since the validation collection differed from the training collection in terms of the source and in terms of the day of processing. For example, the training collection didn't include any patient from the John-Hopkins University. It demonstrates that the phosphosignature can indeed be used for a valid prediction of responsiveness to FLT3 inhibitors.

LMN1 phosphorylation correlates with its protein expression.

Differences in phosphorylation of a specific site may be caused by either a difference in the degree of phosphorylation of this site, a difference in expression of the corresponding protein, or by a combination of both. In order to distinguish between these three possibilities, the proteome in six validation samples was analysed (Fig. 5). For two of the five signature proteins (EEPD1 and LMN1), the phosphorylation and the protein in at least 2/3 of the samples could be quantified. LMN1 shows a very high correlation between phosphorylation and protein expression (Pearson correlation 0.92, p=0.03). The correlation for EEPD1 is smaller and not significantly different from 0 (c=0.70, p=0.18) due to one outlier sample (Fig. 5A). Although the samples were enriched for phosphorylated peptides, non-phosphorylated peptides of LMN1 were identified and quantified in almost all training and validation samples. Therefore, it was possible to correlate the phosphorylation of LMN1 with its expression in these samples (Fig. 5B). A high correlation was obtained (cO.86, p=2.5xlCT⁶).

It has been shown in previous studies that it is feasible to analyse basal activities of signalling pathways to identify markers when analysing cell lines (Andersen, Sathyanarayanan et al. 2010; Klammer, Kaminski et al. 2012). Here, it was demonstrated that the determination of phosphorylation markers directly from clinical is feasible. Global and unbiased quantitative phosphoproteomics was applied to bone marrow samples obtained from AML patients enrolled in a phase II trial of AC220. The methodology was global in the sense, that as many sites as possible were analysed instead of focusing on specific phosphorylation sites. In fact it is unlikely, that any of the identified markers would have been selected as a candidate based on prior knowledge. Despite the small sample amounts of 316 (median protein amount), that can be obtained from bone marrow aspirates, 7,831 class-I phosphorylation sites were detected. 2,1 19 sites were quantified in at least two third of the training samples, so that they could be used for identification of the phospho signature. The methodology was unbiased in the sense, that no subset of phosphorylation events such as tyrosine phosphorylations was selected.

In total pre-treatment bone marrow aspirates from 21 patients enrolled in the AC220 trial were collected. The samples were processed and analysed with a workflow previously tested. Using a first collection of 12 samples, a phospho-signature consisting of five phosphorylation sites was identified. The predictive features were selected by means of the Mean-rank test statistics combined with the ensemble method (Abeel, Helleputte et al. 2010). A SVM with linear kernel was used to train the prediction model. In a first step of evaluating the predictivity of the signature, the classification scheme was cross-validated. The resulting prediction accuracy is 92%, the AUROC 89%. In a second step, the signature was validated with independent nine samples that were not used during training. Seven out of nine patients were correctly classified. One of the misclassified patients (AML033) was classified as responder instead of as non-responder. Actually, the patient's FLT-ITD positive cells were sensitive. However the patient progressed with a FLT3 wild-type clone and wasn't called a responder. Depending on whether this ambiguous call is counted, the resulting accuracy is 78% (with AML033) or 88% (without AML033). In either case, the accuracy is high and in the range of the value determined by cross-validation.

All five phosphorylation sites were identified in previous phosphoproteomic studies. However no function has so far been described for them. For some of the marker proteins relations with AML or cancer in general can be found in the literature. Expression of B-cell lymphoma/leukemia 11 A (BCL11A), for example, is associated with poor outcome of AML patients (Yin, Delwel et al. 2009). Similarly, the expression of Lamins A/C (LMN1) correlated with risk of death from colorectal cancer (Willis, Cox et al. 2008). However, none of the five markers provided herein, particularly B-cell lymphoma/leukemia 1 1A (BCL1 1A), (i.e. neither their phosphorylation status nor their expression) have been described as response prediction marker or response monitoring marker.

These results show the importance of a global and unbiased analysis to enable the identification of markers that have no known association with the drug's main target. For application of the biomarker signature in the clinic it is sufficient to detect and quantify at least one of the five phosphorylation sites. Therefore targeted detection methods, such as immunological methods or the mass-spectrometry-based multiple-reaction-monitoring ( itteringham, Jenkins et al. 2009) can be applied instead. These methods allow reproducible detection and quantification of given peptides from relatively low sample amounts and can be routinely applied to large number of samples.

It was shown that at least one of the marker phosphorylation, LMN1 (S458), strongly correlates with the expression of the corresponding protein. The expression of LMN1 can therefore be used in the alternative to its phosphorylation status as marker for determining whether a proliferative diseased cell or patient responds to an FLT3 inhibitor.

In summary, a global quantitative phosphoproteomics was applied to pre-treatment bone marrow aspirates of AML patients. The proposed signature consisting of at least one of five phosphorylation sites on EEPD1 , BCL1 1A, RANBP3, RP3, LMN1 predicts response to treatment of AML patients with AC220. As we identified the signature directly from clinical samples, a translation of the results from the pre-clinic to the clinic is omitted. Tables

Table 1: Collection of analysed AML samples. CR: complete remission; CRi: CR with insufficient hematological recovery; CRp: CR with insufficient platelet recovery; PR: partial remission; SD; stable disease.

Sample AC220 Protein

Source Patien Id Collection Class

ID Sensitivity [Mg]

AML001 Frankfurt F32446 training n.d. 235

AML002 Frankfurt F32490 training PR + 209

AML003 Frankfurt F32530 training CRp + 386

AML004 Frankfurt F33031 training CRi + 123

AM LOOS Philadelphia 1009-018 training CRi + 800

AML006 Philadelphia 1009-007 training CRi + 230

AML007 Philadelphia 1009-002 training SD - 1020

AML008 Philadelphia 1009-021 training SD - 1720

A L009 Philadelphia 1009-014 training CRi + 456

AML010 Hannover L864VR training SD - 80

AML011 Hannover M212ZM training SD - 108

AML012 Hannover L927C training SD - 72

AML013 Hannover M83BB training SD - 270

AML014 Baltimore 1005-017 validation CRi + 400

AML020 Baltimore 1005-018 validation CRi + 400

AML025 Baltimore 1005-019 validation SD - 350

AML030 Philadelphia 1009-003 validation CRp + 169

AM L031 Philadelphia 1009-009 validation CRi + 459

AML032 Philadelphia 1009-011 validation CRi + 261

AML033 Philadelphia 1009-016 validation SD - 282

AML034 Philadelphia 1009-015 validation NR - 377

A L035 Philadelphia 1009-019 validation CRi + 1254 Table 2: Phosphorylation sites that are significantly different between classes of responder and non- responder. Median diff is the difference of the median log ratios of the responder samples and the median of the non-responder samples.

Median diff

Uniprot id Gene name Site

(LoglO)

Q7L9B9 EEPD1 S160 -1.05

Q9H165 BCL1 1A S630 -0.68

Q9H6Z4 RANBP3 S333 -0.94

Table 3: Phosphorylation sites of the final phospho-signature. Median diff is the difference of the median log ratios of the responder samples and the median of the non-responder samples. SV weight is the weight of the respective feature in the support-vector-machine.

Median diff SV weight

Uniprot id Gene name Site

(LoglO)

Q7L9B9 EEPD1 S160 -1.05 0.75

Q9H165 BCL11A S630 -0.68 0.54

Q9H6Z4 RANBP3 S333 -0.94 0.31

Q92834 RP3 S961 0.64 -0.88

P02545 LMN1 S458 -0.76 0.75

Example 2: Individual phosphorylation markers predict treatment efficacy of neoplasia with a FMS-like tyrosine kinase 3 inhibitor

In Exampe 1, five phosphorylation markers were identified. These phosphorylations (a signature consisting of five phosphorylation sites) were identified as being predictive when used in combination.

Here it was confirmed that also the individual markers correlate with response, too. Fig. 6 shows the normalized ratio of phosphorylation in the respective patient sample compared to the super-SILAC reference for each of the five phosphorylation sites. Though the phosphorylation of the individual proteins leads to more misclassifications than the combined signature, all five marker sites show a clear correlation with response. In particular, phosphorylation of S630 of BCLl 1 A and S333 of RANBP3 lead to only two misclassifications.

Example 3: Correlation of signature sites between bone marrow samples and blood samples

The phospho-signature was identified and validated in bone-marrow samples. Although it is clinical standard procedure to use bone marrow aspirates for diagnosis of AML patients, a predictive test that can be applied to peripheral blood samples would have many advantages. Therefore, we wanted to confirm that the same signature could also be applied to samples from peripheral blood. To this end, peripheral blood samples from three patients before treatment were analyzed, whose bone marrow sample were already used in the validation phase (AML014, AML020, AML025). The peripheral blood samples were processed in the same way as the bone marrow samples. Three markers (BCLl 1 A, EEPD1 and LMN1) show a strong correlation in their phosphorylation in the respective bone marrow and peripheral blood samples (Fig. 7). The correlation coefficient is 0.88 (p=0.009), suggesting that the phosphorylation status of the herein identified markers, such as BCLl 1 A, EEPD1 and LMN1, can be measured and is predictive in both bone marrow and peripheral blood samples.

Example 4: Other phosphorylation sites on the marker proteins

For all five marker proteins additional phosphorylation sites beside the sites included in the phospho-signature of Example 1 were identified. It was confirmed that these phosphorylation sites are also predictive for response to treatment with AC220.

The phosphorylation of the marker sites identified in Example 1 was compared with other sites on the same protein.

For BCLl 1 A, five additional sites (S205, S328, S608, S625, S718) were identified which could be quantified in most of the investigated AML samples. For all sites, the correlation of phosphorylation compared with the marker site S630 was high (in the range 0.69 to 0.95) and significant (p<0.05, see Fig. 8). It is therefore very likely, that not the phosphorylation occupancy differs between responder and non-responder but rather the abundance of the total protein. This suggests that any of these five phosphorylations and/or the expression of BCL11 A can replace the signature site S630.

Similar results were obtained for the EEPDl with the alternative sites S25 and S554. In case of LM 1, we showed in Example 1, that the expression of the total protein can be quantified and that its expression strongly correlates with its phosphorylation at S458.

Example 5: Phosphorylation of BAD

Inhibition of FLT3 leads to a decreased phosphorylation of Bcl-2 antagonist of cell death (BAD) in cell lines with constitutively active FLT3 (Kim, Br J Heamatol. (2006) 134(5):500-9). We were therefore interested to see whether phosphorylation of BAD pre-treatment correlates with response and could therefore be used as predictive marker. We detected and quantified the phosphorylation of site SI 18 on BAD across all investigated AML samples. Yet, the phosphorylation shows no correlation with response (Fig. 9).

The present invention refers to the following nucleotide and amino acid sequences. The sequences provided herein are available in the NCBI database and can be retrieved from world wide web at ncbi .nlm.nih. gov/sites/ entrez?db=gene; Theses sequences also relate to annotated and modified sequences and fragments thereof. The present invention also provides techniques and methods wherein homologous sequences, and variants and fragments of the concise sequences provided herein are used. Preferably, such "variants" are genetic variants.

Seq ID No. 1:

Amino acid sequence of homo sapiens Endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPDl); the phosphorylation site S160 is indicated in bold letters.

>sp I Q7L9B9 I EEPDl_^HUMAN Endonuclease/exonuclease/phosphatase family domain- containing protein 1 OS=Homo sapiens GN=EEPD1 PE=1 SV=2

MGSTLGCHRSIPRDPSDLSHSRKFSAACNFSNILVNQERLNINTATEEELMTLPGVTRAV ARSIVEYREYIGGFKKVEDLALVSGVGATKLEQVKFEICVSSKGSSAQHSPSSLRRDLLA EQQPHHLATAVPLTPRVNIKITATPAQL SVRGLSEKMALSIVDFRREHGPFRSVEDLVRM DGINAAFLDRIRHQVFAERSRPPSTHTNGGLTFTAKPHPSPTSLSLQSEDLDLPPGGPTQ IISTRPSVEAFGGTRDGRPVLRLATWNLQGCSVEKANNPGVREVVC TLLENSIKLLAVQ ELLDREALEKFCTELNQPTLPNIRKW GPRGCWKAVVAEKPSSQLQ GAGYAGFLWDAAA GMELRDAGSQESSPSNGHGKLAGPSPYLGRFKVGSHDLTLVNLHLAALTLLGSENPSKNH SDGHRLASFAQTLQETLKGEKDVIILGDFGQGPDSNDYDILRKEKFHHLIPAHTFTNIST KNPQGSKSLD IWISKSLKKVFTGHWAVVREGLTNP IPDNWS GGVASEHCPVLAEFYT EKDWSKKDAPRNGSGVALERSEANIKHER

In the following amino acid sequence of homo sapiens Endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPD1) phosphorylation sites S25, S160 and S554 are indicated in bold letters.

>sp 1 Q7L9B9 | EEPD1_HUMAN Endonuclease/exonuclease/phosphatase family domain- containing protein 1 OS=Homo sapiens GN=EEPD1 PE=1 SV=2

MGSTLGCHRSIPRDPSDLSHSRKFSAACNFSNILVNQERLNINTATEEELMTLPGVTRAV ARSIVEYREYIGGFKKVEDLALVSGVGATKLEQVKFEICVSSKGSSAQHSPSSLRRDLLA EQQPHHLATAVPLTPRVNINTATPAQLMSVRGLSEK ALSIVDFRREHGPFRSVEDLVRM DGINAAFLDRIRHQVFAERSRPPSTHTNGGLTFTAKPHPSPTSLSLQSEDLDLPPGGPTQ IISTRPSVEAFGGTRDGRPVLRLATWNLQGCSVEKANNPGVREVVCMTLLENSIKLLAVQ ELLDREALEKFCTELNQPTLPNIRKWKGPRGC KAVVAEKPSSQLQKGAGYAGFLWDAAA GMELRDAGSQESSPSNGHGKLAGPSPYLGRFKVGSHDLTLVNLHLAALTLLGSENPSKNH SDGHRLASFAQTLQETLKGEKDVIILGDFGQGPDSNDYDILRKEKFHHLIPAHTFTNIST KNPQGSKSLDNIWISKSLKKVFTGHWAVVREGLTNPWIPDNWSWGGVASEHCPVLAEFYT EKDWSKKDAPRNGSGVALERSEANIKHER

Seq ID No. 2:

Amino acid sequence of homo sapiens B-cell lymphoma leukemia 11A (BCLl lA); the phosphorylation site S630 is indicated in bold letters.

>sp [ Q9H165 I BC11A__HUMAN B-cell lymphoma/leukemia 11A OS=Homo sapiens GN=BCL11A PE=1 SV=2

MSRRKQGKPQHLSKREFSPEPLEAILTDDEPDHGPLGAPEGDHDLLTCGQCQMNFPLGDI LIFIEHKRKQCNGSLCLEKAVDKPPSPSPIEMKKASNPVEVGIQVTPEDDDCLSTSSRGI CPKQEHIADKLLHWRGLSSPRSAHGALIPTPGMSAEYAPQGICKDEPSSYTCTTCKQPFT SAWFLLQHAQNTHGLRIYLESEHGSPLTPRVGIPSGLGAECPSQPPLHGIHIADNNPFNL LRIPGSVSREASGLAEGRFPPTPPLFSPPPRHHLDPHRIERLGAEESi4ALATHHPSAFDRV

LRLNPMAMEPPAMDFSRRLRELAGNTSSPPLSPGRPSPMQRLLQPFQPGSKPPFLATPPL PPLQSAPPPSQPPVKSKSCEFCGKTFKFQSNLVVHRRSHTGEKPYKCNLCDHACTQASKL KRHMKTHMHKSSPMTVKSDDGLSTASSPEPGTSDLVGSASSALKSVVAKFKSENDPNLIP ENGDEEEEEDDEEEEEEEEEEEEELTESERVDYGFGLSLEAARHHENSSRGAVVGVGDES RALPDVMQGMVLSSMQHFSEAFHQVLGEKHKRGHLAEAEGHRDTCDEDSVAGESDRIDDG TVNGRGCSPGESASGGLSKKLLLGSPSSLSPFSKRIKLEKEFDLPPAAMPNTENVYSQ L AGYAASRQLKDPFLSFGDSRQSPFASSSEHSSENGSLRFSTPPGELDGGISGRSGTGSGG STPHISGPGPGRPSSKEGRRSDTCEYCGKVFKNCSNLTVHRRSHTGERPYKCELCNYACA QSSKLTRHMKTHGQVGKDVYKCEICKMPFSVYSTLEKHMKKWHSDRVLNNDIKTE

In the following amino acid sequence of homo sapiens B-cell lymphoma/leukemia 11 A (BCLl 1 A); the phosphorylation sites S205, S328, S608, S625, S630 and S718 are indicated in bold letters.

>sp|Q9Hl65 | BC1 lAJiUMAN B-cell lymphoma/leukemia 11A OS=Homo sapiens GN=BCL11A PE=1 SV=2

MSRRKQGKPQHLSKREFSPEPLEA1LTDDEPDHGPLGAPEGDHDLLTCGQCQMNFPLGDI

LIFIEHKRKQCNGSLCLEKAVDKPPSPSPIEMKKASNPVEVGIQVTPEDDDCLSTSSRGI CPKQEHIADKLLHWRGLSSPRSAHGALIPTPGMSAEYAPQGICKDEPSSYTCTTCKQPFT SAWFLLQHAQNTHGLRIYLESEHGSPLTPRVGIPSGLGAECPSQPPLHGIHIADNNPFNL LRIPGSVSREASGLAEGRFPPTPPLFSPPPRHHLDPHRIERLGAEEMALATHHPSAFDRV LRLNPMAMEPPAMDFSRRLRELAGNTSSPPLSPGRPSPMQRLLQPFQPGSKPPFLATPPL PPLQSAPPPSQPPVKSKSCEFCGKTFKFQSNLVVHRRSHTGEKPYKCNLCDHACTQASKL KRHMKTHMHKSSPMTVKSDDGLSTASSPEPGTSDLVGSASSALKSVVAKFKSENDPNLIP ENGDEEEEEDDEEEEEEEEEEEEELTESERVDYGFGLSLEAARHHENSSRGAVVGVGDES

RALPDVMQGMVLSSMQHFSEAFHQVLGEKHKRGHLAEAEGHRDTCDEDSVAGESDRIDDG TVNGRGCSPGESASGGLSKKLLLGSPSSLSPFSKRIKLEKEFDLPPAAMPNTENVYSQWL AGYAASRQLKDPFLSFGDSRQSPFASSSEHSSENGSLRFSTPPGELDGGISGRSGTGSGG STPHISGPGPGRPSSKEGRRSDTCEYCGKVFKNCSNLTVHRRSHTGERPYKCELCNYACA QSSKLTRHM THGQVGKDVYKCEIC MPFSVYSTLEKHMKKWHSDRVLNNDIKTE

Seq ID No. 3:

Amino acid sequence of homo sapiens Ran-binding protein 3 (RANBP3); the phosphorylation site S333 is indicated in bold letters.

>sp I Q9H6Z4 I ANB3_HUMAN Ran-binding protein 3 OS=Homo sapiens GN=RANBP3 PE=1 SV=1

MADLANEEKPAIAPPVFVFQKDKGQKSPAEQKNLSDSGEEPRGEAEAPHHGTGHPESAGE

HALEPPAPAGASASTPPPPAPEAQLPPFPRELAGRSAGGSSPEGGEDSDREDGNYCPPVK

RERTSSLTQFPPSQSEERSSGFRLKPPTLIHGQAPSAGLPSQKPKEQQRSVLRPAVLQAP

QPKALSQTVPSSGTNGVSLPADCTGAVPAASPDTAAWRSPSEAADEVCALEEKEPQKNES

SNASEEEACEKKDPATQQAFVFGQNLRDRVKLINESVDEADMENAGHPSADTPTATNYFL

QYISSSLENSTNSADASSNKFVFGQNMSERVLSPPKLNEVSSDANRENAAAESGSESSSQ

EATPEKESLAESAAAYTKATARKCLLEKVEVITGEEAES VLQMQCKLFVFDKTSQSWVE

RGRGLLRLNDMASTDDGTLQSRLVMRTQGSLRLILNTKL AQMQIDKASEKSI ITAMDT

EDQGVKVFLISASSKDTGQLYAALHHRILALRSRVEQEQEAKMPAPEPGAAPSNEEDDSD

DDDVLAPSGATAAGAGDEGDGQTTGST

Seq ID No. 4:

Amino acid sequence of homo sapiens X-linked retinitis pigmentosa GTPase regulator (RP3); the phosphorylation site S961 is indicated in bold letters.

>sp| Q92834 | RPGR_^HUMAN X-linked retinitis pigmentosa GTPase regulator OS=Homo sapiens GN=RPGR PE=1 SV=2

MREPEELMPDSGAVFTFGKSKFAENNPGKFWFKNDVPVHLSCGDEHSAVVTGNNKLYMFG SNNWGQLGLGSKSAISKPTCVKALKPEKVKLAACGRNHTLVSTEGGNVYATGGNNEGQLG LGDTEERNTFHVISFFTSEHKIKQLSAGSNTSAALTEDGRLFMWGDNSEGQIGLKNVSNV CVPQQVTIGKPVSWISCGYYHSAFVTTDGELYVFGEPENGKLGLPNQLLGNHRTPQLVSE IPEKVIQVACGGEHTWLTENAVYTFGLGQFGQLGLGTFLFETSEPKVIENIRDQTISYI SCGENHTALITDIGLMYTFGDGRHGKLGLGLENFTNHFIPTLCSNFLRFIVKLVACGGCH MVVFAAPHRGVA EIEFDEINDTCLSVATFLPYSSLTSGNVLQRTLSARMRRRERERSPD SFSMRRTLPPIEGTLGLSACFLPNSVFPRCSERNLQESVLSEQDLMQPEEPDYLLDE TK EAEIDNSSTVESLGETTDILNMTHIMSLNSNEKSLKLSPVQKQKKQQTIGELTQDTALTE NDDSDEYEEMSEM EGKACKQHVSQGIFMTQPATTIEAFSDEEVGNDTGQVGPQADTDGE GLQKEVYRHENNNGVDQLDAKEIEKESDGGHSQKESEAEEIDSE ETKLAEIAGMKDLRE REKSTKKMSPFFGNLPDRGMNTESEENKDFVKKRESCKQDVIFDSERESVEKPDSYMEGA SESQQGIADGFQQPEAIEFSSGEKEDDEVETDQNIRYGRKLIEQGNEKETKPIISKSMAK YDFKCDRLSEIPEE EGAEDSKGNGIEEQEVEANEENVKVHGGRKEKTEILSDDLTDKAE DHEFSKTEELKLEDVDEEINAENVESKKKTVGDDESVPTGYHSKTEGAERTNDDSSAETI E KEKANLEERAICEYNENPKGYMLDDADSSSLEILENSETTPSKDMKKTKKIFLF RVP SINQKIV NNNEPLPEIKSIGDQIILKSDNKDADQNHMSQNHQNIPPTNTERRSKSCTIL

Seq ID No. 5:

Amino acid sequence of homo sapiens Lamin-A/C (LMNl); the phosphorylation site S458 is indicated in bold letters. >sp I P02545 I LMNA_^HUMAN Prelamin-A/C OS=Homo sapiens GN=LMNA PE

METPSQRRATRSGAQASSTPLSPTRITRLQE EDLQELNDRLAVYIDRVRSLETENAGLR

LRITESEEVVSREVSGI AAYEAELGDARKTLDSVAKERARLQLELSKVREEFKELKARN

TKKEGDLIAAQARLKDLEALLNSKEAALSTALSE RTLEGELHDLRGQVAKLEAALGEAK

KQLQDEMLRRVDAENRLQTMKEELDFQKNIYSEELRETKRRHETRLVEIDNGKQREFESR

LADALQELRAQHEDQVEQYK ELE TYSAKLDNARQSAERNSNLVGAAHEELQQSRIRID

SLSAQLSQLQKQLAAKEAKLRDLEDSLARERDTSRRLLAEKEREMAEMRARMQQQLDEYQ

ELLDIKLALD EIHAYRKLLEGEEERLRLSPSPTSQRSRGRASSHSSQTQGGGSVTK RK

LESTESRSSFSQHARTSGRVAVEEVDEEGKFVRLRNKSNEDQSMGNWQI RQNGDDPLLT

YRFPP FTLKAGQVVTIWAAGAGATHSPPTDLVWKAQNTWGCGNSLRTALINSTGEEVAM

RKLVRSVTVVEDDEDEDGDDLLHHHHGSHCSSSGDPAEYNLRSRTVLCGTCGQPADKASA

SGSGAQVGGPISSGSSASSVTVTRSYRSVGGSGGGSFGDNLVTRSYLLGNSSPRTQSPQN

CSIM

Seq ID No. 6:

Nucleotide sequence encoding homo sapiens Prelainin A/C (LMNA) (corresponds to Uniprot id P02545).

>gi I 383792147 ] ref 1 NM_170707.3 | Homo sapiens lamin A/C (LMNA), transcript

1, mRNA

AGGAGGACCTAITAGAGCCTTTGCCCCGGCGTCGGTGACTCAGTGTTCGCGGGAGCGCCGCACCTACACC AGCCAACCCAGATCCCGAGGTCCGACAGCGCCCGGCCCAGATCCCCACGCCTGCCAGGAGCAAGCCGAGA GCCAGCCGGCCGGCGCACTCCGACTCCGAGCAGTCTCTGTCCTTCGACCCGAGCCCCGCGCCCTTTCCGG GACCCCTGCCCCGCGGGCAGCGCTGCCAACCTGCCGGCCATGGAGACCCCGTCCCAGCGGCGCGCCACCC GCAGCGGGGCGCAGGCCAGCTCCACTCCGCTGTCGCCCACCCGCATCACCCGGCTGCAGGAGAAGGAGGA CCTGCAGGAGCTCAATGATCGCTTGGCGGTCTACATCGACCGTGTGCGCTCGCTGGAAACGGAGAACGCA GGGCTGCGCCTTCGCATCACCGAGTCTGAAGAGGTGGTCAGCCGCGAGGTGTCCGGCATCAAGGCCGCCT ACGAGGCCGAGCTCGGGGATGCCCGCAAGACCCTTGACTCAGTAGCCAAGGAGCGCGCCCGCCTGCAGCT GGAGCTGAGCAAAGTGCGTGAGGAGTTTAAGGAGCTGAAAGCGCGCAATACCAAGAAGGAGGGTGACCTG ATAGCTGCTCAGGCTCGGCTGAAGGACCTGGAGGCTCTGCTGAACTCCAAGGAGGCCGCACTGAGCACTG CTCTCAGTGAGAAGCGCACGCTGGAGGGCGAGCTGCATGATCTGCGGGGCCAGGTGGCCAAGCTTGAGGC AGCCCTAGGTGAGGCCAAGAAGCAACTTCAGGATGAGATGCTGCGGCGGGTGGATGCTGAGAACAGGCTG CAGACCATGAAGGAGGAACTGGACTTCCAGAAGAACATCTACAGTGAGGAGCTGCGTGAGACCAAGCGCC GTCATGAGACCCGACTGGTGGAGATTGACAATGGGAAGCAGCGTGAGTTTGAGAGCCGGCTGGCGGATGC GCTGCAGGAACTGCGGGCCCAGCATGAGGACCAGGTGGAGCAGTATAAGAAGGAGCTGGAGAAGACTTAT TCTGCCAAGCTGGACAATGCCAGGCAGTCTGCTGAGAGGAACAGCAACCTGGTGGGGGCTGCCCACGAGG AGCTGCAGCAGTCGCGCATCCGCATCGACAGCCTCTCTGCCCAGCTCAGCCAGCTCCAGAAGCAGCTGGC AGCCAAGGAGGCGAAGCTTCGAGACCTGGAGGACTCACTGGCCCGTGAGCGGGACACCAGCCGGCGGCTG CTGGCGGAAAAGGAGCGGGAGATGGCCGAGATGCGGGCAAGGATGCAGCAGCAGCTGGACGAGTACCAGG AGCTTCTGGACATCAAGCTGGCCCTGGACATGGAGATCCACGCCTACCGCAAGCTCTTGGAGGGCGAGGA GGAGAGGCTACGCCTGTCCCCCAGCCCTACCTCGCAGCGCAGCCGTGGCCGTGCTTCCTCTCACTCATCC CAGACACAGGGTGGGGGCAGCGTCACCAAAAAGCGCAAACTGGAGTCCACTGAGAGCCGCAGCAGCTTCT CACAGCACGCACGCACTAGCGGGCGCGTGGCCGTGGAGGAGGTGGATGAGGAGGGCAAGTTTGTCCGGCT GCGCAACAAGTCCAATGAGGACCAGTCCATGGGCAATTGGCAGATCAAGCGCCAGAATGGAGATGATCCC TTGCTGACTTACCGGTTCCCACCAAAGTTCACCCTGAAGGCTGGGCAGGTGGTGACGATCTGGGCTGCAG GAGCTGGGGCCACCCACAGCCCCCCTACCGACCTGGTGTGGAAGGCACAGAACACCTGGGGCTGCGGGAA CAGCCTGCGTACGGCTCTCATCAACTCCACTGGGGAAGAAGTGGCCATGCGCAAGCTGGTGCGCTCAGTG ACTGTGGTTGAGGACGACGAGGATGAGGATGGAGATGACCTGCTCCATCACCACCACGGCTCCCACTGCA GCAGCTCGGGGGACCCCGCTGAGTACAACCTGCGCTCGCGCACCGTGCTGTGCGGGACCTGCGGGCAGCC TGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGCGGACCCATCTCCTCTGGCTCTTCTGCC TCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGGGGTGGCAGCTTCGGGGACAATC TGGTCACCCGCTCCTACCTCCTGGGCAACTCCAGCCCCCGAACCCAGAGCCCCCAGAACTGCAGCATCAT GTAATCTGGGACCTGCCAGGCAGGGGTGGGGGTGGAGGCTTCCTGCGTCCTCCTCACCTCATGCCCACCC CCTGCCCTGCACGTCATGGGAGGGGGCTTGAAGCCAAAGAAAAATAACCCTTTGGTTTTTTTCTTCTGTA TTTTTTTTTCTAAGAGAAGT ATTTTCTACAGTGGTTTTATACTGAAGGAAAAACACAAGCAAAAAAAAA AAAAAGCATCTATCTCATCTATCTCAATCCTAATTTCTCCTCCCTTCCTTTTCCCTGCTTCCAGGAAACT CCACATCTGCCTTAAAACCAAAGAGGGCTTCCTCTAGAAGCCAAGGGAAAGGGGTGCTTTTATAGAGGCT AGCTTCTGCTTTTCTGCCCTGGCTGCTGCCCCCACCCCGGGGACCCTGTGACATGGTGCCTGAGAGGCAG GCATAGAGGCTTCTCCGCCAGCCTCCTCTGGACGGCAGGCTCACTGCCAGGCCAGCCTCCGAGAGGGAGA GAGAGAGAGAGAGGACAGCTTGAGCCGGGCCCCTGGGCTTGGCCTGCTGTGATTCCACTACACCTGGCTG AGGTTCCTCTGCCTGCCCCGCCCCCAGTCCCCACCCCTGCCCCCAGCCCCGGGGTGAGTCCATTCTCCCA GGTACCAGCTGCGCTTGCTTTTCTGTATTTTATTTAGACAAGAGATGGGAATGAGGTGGGAGGTGGAAGA AGGGAGAAGAAAGGTGAGTTTGAGCTGCCTTCCCTAGCTTTAGACCCTGGGTGGGCTCTGTGCAGTCACT GGAGGTTGAAGCCAAGTGGGGTGCTGGGAGGAGGGAGAGGGAGGTCACTGGAAAGGGGAGAGCCTGCTGG CACCCACCGTGGAGGAGGAAGGCAAGAGGGGGTGGAGGGGTGTGGCAGTGGTTTTGGCAAACGCTAAAGA GCCCTTGCCTCCCCATTTCCCATCTGCACCCCTTCTCTCCTCCCCAAATCAATACACTAGTTGTTTCTAC CCCTGGCAAAAAAAAAAAA

Seq ID No. 7:

Amino acid sequence of FMS-like tyrosine kinase 3 (FLT3).

>sp| P36888 t FLT3__HUMAN Receptor-type tyrosine-protein kinase FLT3 OS=Homo sapiens GN=FLT3 PE=1 SV=2

MPALARDGGQLPLLVVFSAMIFGTITNQDLPVI CVLINHKNNDSSVGKSSSYPMVSESP EDLGCALRPQSSGTVYEAAAVEVDVSASITLQVLVDAPGNISCLWVF HSSLNCQPHFDL QNRGVVSMVILKMTETQAGEYLLFIQSEATNYTILFTVSIRNTLLYTLRRPYFRKMENQD ALVCISESVPEPIVE VLCDSQGESCKEESPAVVKKEEKVLHELFGTDIRCCARNELGRE CTRLFTIDLNQTPQTTLPQLFLKVGEPL IRCKAVHVNHGFGLTWELENKALEEGNYFEM STYSTNRTMIRILFAFVSSVARNDTGYYTCSSSKHPSQSALVTIVEKGFINATNSSEDYE IDQYEEFCFSVRFKAYPQIRCTWTFSR SFPCEQKGLDNGYSISKFCNHKHQPGEYIFHA ENDDAQFT MFTLNIRRKPQVLAEASASQASCFSDGYPLPS TWKKCSDKSPNCTEEITE GVWNR ANRKVFGQWVSSSTLNMSEAIKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQD NISFYATIGVCLLFIVVLTLLICH YKKQFRYESQLQMVQVTGSSDNEYFYVDFREYEYD LKWEFPRENLEFGKVLGSGAFGKVMNATAYGIS TGVSIQVAV MLKEKADSSEREALMS ELKMMTQLGSHENIVNLLGACTLSGPIYLI FEYCCYGDLLNYLRSKREKFHRTWTEIFKE HNFSFYPTFQSHPNSSMPGSREVQIHPDSDQISGLHGNSFHSEDEIEYENQKRLEEEEDL NVLTFEDLLCFAYQVAKGMEFLEFKSCVHRDLAARNVLVTHGKVVKICDFGLARDIMSDS NYVVRGNARLPVKWMAPESLFEGIY IKSDVWSYGILLWEIFSLGVNPYPGIPVDANFYK LIQ GFKMDQPFYATEEIYII QSC AFDSRKRPSFPNLTSFLGCQLADAEEAMYQNVDG RVSECPHTYQNRRPFSREMDLGLLSPQAQVEDS

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Claims

1. A method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of one or more of B-cell lymphoma/leukemia 1 1A protein (BCL1 1A), Lamin AJC (LMN1 ), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl ), Ran-binding protein 3 (RANBP3) and/or GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

2. A method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPDl) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

3. The method of claim 2, further comprising determining the phosphorylation status of one or more of B-cell lymphoma/leukemia 1 1A protein (BCL1 1A), Ran-binding protein 3 (RANBP3), GTPase regulator (RP3) and/or Lamin A/C (LMN1) in a sample of a patient, wherein said status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

4. A method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of B-cell lymphoma/leukemia 1 1A protein (BCL1 1A) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

5. The method of claim 4, further comprising determining the phosphorylation status of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), Ran-binding protein 3 (RANBP3), GTPase regulator (RP3) and/or Lamin A/C (LMN1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

6. A method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Ran-binding protein 3 (RANBP3), in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

7. The method of claim 6, further comprising determining the phosphorylation status of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), B-ceil lymphoma/leukemia 1 1A protein (BCL1 1 A), GTPase regulator (RP3) and/or Lamin A/C (LMN1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

8. A method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

9. The method of claim 8, further comprising determining the phosphorylation status of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), B-cell lymphoma/leukemia 11A protein (BCL11A), Ran-binding protein 3 (RANBP3), and/or Lamin A/C (LMN1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

10. A method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the phosphorylation status of Lamin A/C (LMN1) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

11. The method of claim 10, further comprising detenriining the phosphorylation status of one or more of endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), B-cell lymphoma/leukemia 1 1A protein (BCL1 1A), Ran-binding protein 3 (RANBP3), and/or GTPase regulator (RP3) in a sample of a patient, wherein said phosphorylation status is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

12. The method of any one of claims 1 to 1 1 , wherein said phosphorylation status is the presence or absence of phosporylation at one or more phosphorylation sites of one or more of B-cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), Ran- binding protein 3 (RANBP3), and/or GTPase regulator (RP3).

13. The method of claim 12, wherein the absence of phosporylation at one or more phosphorylation sites of one or more of B-cell lymphoma/leukemia 11A protein (BCL1 1 A), Lamin A C (LMNl), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPDl), and/or Ran-binding protein 3 (RANBP3) is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

14. The method of claim 12 or 13, wherein the presence of phosporylation at one or more phosphorylation sites of GTPase regulator (RP3) is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

15. The method of any one of claims 1 to 1 1, wherein a decrease of phosphorylated phosphorylation sites of one or more of B-cell lymphoma/leukemia 11 A protein (BCL11A), Lamin A/C (LMNl), endonuclease/exonuclease/phosphatase family domain- containing protein 1 (EEPDl), and/or Ran-binding protein 3 (RANBP3), in comparison to a control is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor and/or wherein an increase of phosphorylated phosphorylation sites of GTPase regulator (RP3) in comparison to a control is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

16. The method of any one of claims 12 to 15, wherein said phosphorylation site is one or more of the following phosphorylation sites:

a. S160 of EEPDl;

b. S630 ofBCLl lA;

c. S333 of RANBP3;

d. S961 of RP3; and/or

e. S458 of LMNl .

17. The method of any one of claims 12 to 16, wherein the presence or absence of phosphorylation of the combination of phosphorylation sites SI 60 of EEPDl ; S630 of BCL11A; S333 of RANBP3; S961 ofRP3; and S458 of LMN1 is to be determined; or the method of claim 15 or 16, wherein an increase or decrease of phosphorylated phosphorylation sites of the combination of phosphorylation sites SI 60 of EEPDl ; S630 of BCL1 1 A; S333 of RANBP3; S961 of RP3; and S458 of LMN 1 is to be determined.

18. The method of any one of claims 1 to 17, further comprising determining the expression level of Lamin A/C (LMN1).

19. The method of claim 18, wherein a decrease in said expression level in comparison to the control is indicative of the responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

20. A method of determining whether a proliferative diseased cell is responsive to an FMS-like tyrosine kinase 3 (FLT3) inhibitor, said method comprising determining the expression level of Lamin A/C (LMNl) in a sample of a patient, wherein said expression level is indicative of responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

21. The method of claim 20, wherein a decrease in said expression level in comparison to the control is indicative of the responsiveness to said FMS-like tyrosine kinase 3 (FLT3) inhibitor.

22. The method of any one of claims 18 to 21, wherein the expression level of Lamin A/C (LMNl) is at least 2.5-fold, preferably at least 5-fold decreased in comparison to the control.

23. The method of any one of claims 18 to 22, wherein the expression level of Lamin A/C (LMN1) is the mRNA expression level of Lamin A/C (LMN1).

24. The method of claim 23, wherein the mRNA expression level is assessed by in situ hybridization, micro-arrays, or RealTime PCR.

25. The method of any one of claims 18 to 22, wherein the expression level of Lamin A/C (LMNl) is the protein expression level of Lamin A/C (LM 1).

26. The method of claim 25, wherein said protein expression level is assessed by immunoassay, gel- or blot-based methods, IHC, mass spectrometry, flow cytometry, or FACS.

27. The method of any one of claims 1 to 26, wherein said phosphorylation is detected by immunoassay, IHC, mass spectrometry or intracellular flow cytometry.

28. The method of any one of claims 1 to 27, wherein said FMS-like tyrosine kinase 3 (FLT3) inhibitor is selected from the group consisting of quizartinib (AC220), midostaurin (P C- 412), lestaurinib (CEP-701), 4SC-203, sorafenib (Bay-43-0006), sunitinib (SU-1 1248), and tandutinib (MLN-0518).

29. The method of any one of claims 1 to 27, wherein said FMS-like tyrosine kinase 3 (FLT3) inhibitor is quizartinib (AC220).

30. The method of any one of claims 1 to 27, wherein said inhibitor is a selective FMS-like tyrosine kinase 3 (FLT3) inhibitor.

31. The method of any one of claims 1 to 30, wherein the patient is suspected to suffer from neoplasia, suffering from neoplasia or being prone to suffer from neoplasia.

32. The method of claim 31 , wherein said neoplasia is a malignant neoplasia.

33. The method of claim 32, wherein said malignant neoplasia is leukemia.

34. The method of claim 33, wherein said leukemia is myeloid leukemia.

35. The method of claim 34, wherein said myeloid leukemia is acute myeloid leukemia (AML).

36. The method of claim 33, wherein said leukemia is lymphoid leukemia.

37. The method of claim 36, wherein said lymphoid leukemia is acute lymphoid leukemia (ALL).

38. The method of claim 31 , wherein said neoplasia is a myelodysplastic syndrome.

39. The method of claim 38, wherein said myelodysplastic syndrome is refractory anemia with excess of blasts (RAEB I or RAEB II).

40. The method of any one of claims 1 to 39, wherein an FMS-like tyrosine kinase 3 (FLT3) inhibitor is to be administered to the patient.

41. A method of treating a patient, said method comprising selecting a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is determined

(i) to have one or more of B-cell lymphoma leukemia 11A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1) and/or Ran-binding protein 3 (RANBP3) not phosphorylated; and/or

(ii) to have GTPase regulator (RP3) phosphorylated; and

42. A method of treating a patient, said method comprising selecting a neoplasia patient, wherein proliferative diseased cells of a sample of the patient are determined,

(i) to have a decrease of phosphorylated phosphorylation sites of one or more of B-cell lymphoma/leukemia 1 1A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), and/or Ran-binding protein 3 (RANBP3), in comparison to a control and/or

(ii) to have an increase of phosphorylated phosphorylation sites of GTPase regulator (RP3) in comparison to a control; and

43. An FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient

(i) is not phosphorylated at one or more phosphorylation sites of one or more of B-cell lymphoma/leukemia 1 1A protein (BCL11A), Lamin A/C (LM 1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), and/or Ran-binding protein 3 (RANBP3); and/or

44 An FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein proliferative diseased cells of a sample of the patient

(i) are determined to have a decrease of phosphorylated phosphorylation sites of one or more of B-cell lymphoma/leukemia 11A protein (BCL11A), Lamin A/C (LMN1), endonuclease/exonuclease/phosphatase family domain-containing protein 1 (EEPD1), and/or Ran-binding protein 3 (RANBP3), in comparison to a control; and/or

(ii) are determined to have an increase of phosphorylated phosphorylation sites of GTPase regulator (RP3) in comparison to a control.

45. The method of claim 41 or 42, or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 43 or 44, wherein said phosphorylation site is one or more of the following phosphorylation sites:

a. SI 60 of EEPD1 ;

b. S630 of BCLl lA;

c. S333 of RANBP3;

d. S961 of RP3; and/or

e. S458 ofLMNl .

46. The method of claim 41 or 45, wherein said proliferative diseased cell is determined to have the phosphorylation sites S160 of EEPDl; S630 of BCLl lA; S333 of RANBP3; S458 of LMN1 not phosphorylated; and wherein said proliferative diseased cell is determined to have the phosphorylation site S961 of RP3 phosphorylated; or

the method of claim 42 or 45, wherein said said proliferative diseased cells are determined to have a decrease of phosphorylated phosphorylation sites S160 of EEPDl ; S630 of BCL11 A; S333 of RANBP3; S458 of LMN1 in comparison to a control and/or to have an increase of phosphorylated phosphorylation site S961 of GTPase regulator (RP3) in comparison to a control.

47. The FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 43 or 45, wherein the phosphorylation sites S160 of EEPDl ; S630 of BCLl lA; S333 of RANBP3; and S458 of LMN1 are not phosphorylated; and wherein the phosphorylation site S961 of RP3 is phosphorylated; or

the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 44, wherein said proliferative diseased cells are determined to have a decrease of phosphorylated phosphorylation sites of the phosphorylation sites S160 of EEPDl ; S630 of BCLl lA; S333 of RANBP3; S458 of LMN1 in comparison to a control and/or to have an increase of phosphorylated phosphorylation site S961 of GTPase regulator (RP3) in comparison to a control.

48. The method of any one of claims 41, 42, 45 and 46„ wherein said proliferative diseased cell is determined to have Lamin A/C (LMN1) expressed at a decreased level in comparison to the control.

49. The FMS-like tyrosine kinase 3 (FLT3) inhibitor of any one of claims 43, 44, 45 and 47, , wherein said proliferative diseased cell is characterized by Lamin A/C (LMN1) expression.

50. A method of treating a patient, said method comprising selecting a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is determined to have Lamin A/C (LMN1) expressed; and administering to the patient an effective amount of an FMS- like tyrosine kinase 3 (FLT3) inhibitor.

51. An FMS-like tyrosine kinase 3 (FLT3) inhibitor for use in treating a neoplasia patient, wherein a proliferative diseased cell of a sample of the patient is characterized by Lamin A/C (LMN1) expression

52. The method of claim 48 or 50; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 49 or 51, wherein the expression level of Lamin A/C (LM l ) is decreased in comparison to the control.

53. The method of claim 48 or 52; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 51 or 52, wherein the expression level of Lamin A/C (LMNl) is at least 2.5-fold, preferably at least 5-fold decreased in comparison to the control.

54. The method of any one of one of claims 41, 42, 45, 46, 48, 50 52 and 53; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of any one of claims43, 44, 45, 47, 49, 51 to 53, wherein said FMS-like tyrosine kinase 3 (FLT3) inhibitor is selected from the group consisting of quizartinib (AC220), midostaurin (P C-412), lestaurinib (CEP-701 ), 4SC-203, sorafenib (Bay-43-0006), sunitinib (SU-11248), and tandutinib (MLN-0518).

55. The method of any one of one of claims 41, 42, 45, 46, 48, 50, 52 and 53; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of any one of claims 43, 44, 45, 47, 49, 51 to 53, wherein said FMS-like tyrosine kinase 3 (FLT3) inhibitor is quizartinib (AC220).

56. The method of any one of one of 41, 42, 45, 46, 48, 50, 52 and 53; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of any one of claims 43, 44, 45, 47, 49, 51 to 53, wherein said inhibitor is a selective FMS-like tyrosine kinase 3 (FLT3) inhibitor.

57. The method of any one of one of claims 41, 42, 45, 46, 48, 50, and 52 to 56; or the FMS- like tyrosine kinase 3 (FLT3) inhibitor of any one of claims 43, 44, 45, 47, 49, 51 to 56, wherein the patient is suspected to suffer from neoplasia, suffering from neoplasia or being prone to suffer from neoplasia.

58. The method of claim 57; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 57, wherein said neoplasia is a malignant neoplasia.

59. The method of claim 58; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 58, wherein said malignant neoplasia is leukemia.

60. The method of claim 59; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 59, wherein said leukemia is myeloid leukemia.

61. The method of claim 60; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 60, wherein said myeloid leukemia is acute myeloid leukemia (AML).

62. The method of claim 59; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 59, wherein said leukemia is lymphoid leukemia.

63. The method of claim 62; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 62, wherein said lymphoid leukemia is acute lymphoid leukemia (ALL).

64. The method of claim 57; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 57, wherein said neoplasia is a myelodysplasia syndrome.

65. The method of claim 64; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of claim 64, wherein said myelodysplastic syndrome is refractory anemia with excess of blasts (RAEB I or RAEB II).

66. The method of any one of one of claims 1 to 42, 45, 46, 48, 50, 52 to 65; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of any one of claims 43, 44, 45, 47, 49, 51 to 66, wherein said sample is a bone marrow sample.

67. The method of any one of one of claims 1 to 42, 45, 46, 48, 50, 52 to 65; or the FMS-like tyrosine kinase 3 (FLT3) inhibitor of any one of claims 43, 44, 45, 47, 49, 51 to 66, wherein said sample is a blood sample, in particular a peripheral blood sample.