AMPK PATHWAY AS A THERANOSTIC INDICATOR FOR MIXED

LINEAGE LEUKEMIA (MLL)

BACKGROUND INFORMATION Acute lymphoblastic leukemias (ALL), which primarily affect children and young adults, can be sub-divided into three major classes: B-ALL (B-cell acute lymphoblastic leukemia), T- ALL (T-cell acute lymphoblastic leukemia) and MLL (mixed lineage leukemias, which include myeloid/lymphoid leukemias). Unlike the majority of childhood ALL, subjects with MLL exhibit I lq23 chromosomal rearrangements, and exhibit an especially high risk of treatment failure and poor prognosis.

The development of effective therapy for children with some forms of ALL has greatly improved long-term event-free survival rates, but 30% of patients are still not able to reach a stable complete remission. Infants with MLL form the most striking example of ALL patients who do not benefit from the improved treatments (4-yr EFS 40%). The failure of hematopoietic stem cell transplantation to improve clinical outcome for the MLL subgroup suggests that greater emphasis should be placed on the eradication of residual drug-resistant leukemic cells, the source of most treatment failure in these patients. It would be desirable to identify new therapeutic targets for treating such patients.

DESCRIPTION OF THE DRAWINGS

Figure 1 schematically illustrates two types of assays. Fig. IA shows a forward phase array, in which a bait molecule, such as an antibody designed to capture specific analytes, is immobilized and reacted with a mixture of test sample proteins. Fig. IB shows a reverse phase protein array (RPPA) in which test samples (e.g., cell lysates) containing analytes of interest are immobilized on a solid phase, and an analyte specific ligand (e.g., an antibody) is applied in solution phase. See, e.g., Liotta et al. (2003) Cancer Cell 3, 317-25.

Figure 2 shows an example of a stained Reverse Phase Protein Array from this study.

Figure 3 shows RPPA Results. Fig. 3A shows diagrammatically that proteins of the AMP- activated protein kinase (AMPK) pathway are significantly more activated in MLL rearranged patients than in B-ALL patients. Figure 3B is a histogram summarizing these findings, showing means and standard deviations.

Figures 4A and 4B show a cytofluorimetric example of apoptotic induction by the AMPK inhibitor, (6-[4-(2-Piperidin-l-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrr azolo[l,5-a]-pyrimidine, in RS4;11 (Fig. 4A) and REH (Fig. 4B) cells. Figure 4C shows viable cells after treatment with the AMPK inhibitor, normalized on the control (untreated cells).

Figures 5A and 5B show a cytofluorimetric example of apoptotic induction by the ERK 1/2 inhibitor in RS4;1 1 (Fig. 5A) and REH (Fig. 5B) cells. Figure 5C shows viable cells after treatment with the ERK 1/2 inhibitor, normalized on the control (untreated cells).

DESCRIPTION OF THE INVENTION

The present inventors have measured the activation of signal transduction pathways (STPs) in subjects having MLL and have identified a signal transduction pathway - the AMPK pathway - members (proteins) of which are aberrantly activated (the proteins are significantly more highly phosphorylated) in subjects having MLL (with the rearranged chromosomal abnormality) than in subjects having B-ALL (lacking the translocation). These activated proteins within the AMPK signaling pathway can serve as molecular targets for therapy.

Advantageously, this new set of targets provides a method for treating a disease that was heretofore essentially untreatable. A method of the invention for detecting a class of MLL subjects that is likely to be responsive to treatment with an inhibitor of one or members of the AMPK pathway is rapid, accurate and inexpensive.

One aspect of the invention is a method for identifying a class of subjects (e.g., human patients) having MLL who are likely to be responsive (susceptible) to treatment with an inhibitor of a member of the AMPK signaling pathway. The method comprises measuring, in a sample from the subject, the amount of phosphorylation of (a) LKBl at residue S428, (b) AMPKα at residue S485, (c) AMPKβ at residue S 108, and/or (d) eNOS III at residue Sl 16. The method may comprise measuring the total amount of LKBl, AMPKα, AMPKβ and/or eNOS III that is phosphorylated at the indicated residues (e.g. the amount of each phosphoprotein in the sample). It should be understood that a discussion herein of the former method (measuring the amount of phosphorylation ...), as used herein, is also meant to encompass the latter method of measurement (measuring the total amount of the phosphoprotein isoform). An elevated level (e.g. a significantly elevated level) of phosphorylation at one or more (e.g., one, two, three or all

four) of these residues, compared to a baseline value or reference standard (e.g., a negative reference standard), for example by comparison to calibration curve, indicates that the subject is likely to be responsive to therapy with an inhibitor of a member of the AMPK pathway.

In one embodiment of the invention, the amount of activation (phosphorylation) of one or more (e.g., at least about 5, 10, 25, 50, 75 or 100 or more) members of the AMPK signaling pathway or interconnected pathways may be determined, instead of or addition to the four members of the pathway which are discussed above. A variety of phosphoproteins are connected to and/or emanate from the pathway with four elements that is illustrated in Figure 3A. Such phosphoproteins/ signaling pathways will be evident to a skilled worker. They include, e.g., TORC2, ACC, mTOR, AKT, 4EBP1, HMG Co-A reductase, GSK3 and p70S6K and related pathway members.

In one embodiment of the invention, subjects having MLL are subdivided into two classes, those which are likely to be responsive to the treatment, and those which are not.

As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, "an" inhibitor, as used above, includes two or more such inhibitors of a given target, or two or more inhibitors of different targets; and "a" member of a pathway includes one or more members of the pathway, individually or in combination.

Another aspect of the invention is a method of identification as above, which further comprises administering to a subject which is shown to be likely to be responsive to treatment with an inhibitor of a member of the AMPK signaling pathway, an effective amount of an agent (e.g. a combination of agents) that inhibits the amount or activity of a phosphorylated form of a member of the AMPK pathway (e.g., of LKBl, AMPKα, AMPKβ and/or eNOS III), or of one or more (e.g., at least about 5, 10, 25, 50, 75 or 100) additional members of the AMPK pathway, individually or in combination. The term "inhibitor," as used herein, refers to an agent that can inhibit the amount and/or or the activity of a phosphorylated protein of the invention. As used herein, agents that inhibit the "amount" of a phosphorylated protein of the invention can inhibit the expression of the protein, thereby reducing the total amount of the protein, or can inhibit the phosphorylation of the protein, thereby reducing the total amount of the phosphorylated protein. Another aspect of the invention is method for treating a subject having MLL, comprising administering to the subject an effective amount of an agent (e.g. a combination of agents) that inhibits the amount or activity of a phosphorylated form of a member of the AMPK signaling

pathway (e.g., LKBl, AMPKα, AMPKβ and/or eNOS III). One class of subjects that can be given this therapy is a class of MLL subjects from which a sample is shown to exhibit a significantly elevated level of phosphorylation at one or more particular sites of one or more members of the AMPK signaling pathway (e.g., at residues S428 of LKBl, S485 of AMPKα, S 108 of AMPKβ, and/or S 1 16 of eNOS III), compared to a baseline value.

Another aspect of the invention is in a method for treating a subject having MLL, the improvement comprising determining that a sample from the subject exhibits a significantly elevated level of phosphorylation at residue S428 of LKBl, residue S485 of AMPKα, residue S108 of AMPKβ, and/or residue Sl 16 of eNOS III, compared to a baseline, and then administering an effective amount of an agent (e.g., a combination of agents) that inhibits the amount or activity of the phosphorylated form of a member of the AMPK pathway.

In one aspect of the invention, the amount of phosphorylation at one or more of the noted residues is detected by measuring the amount of reactivity of an antibody specific for the phosphorylated isoform of the residue in a sample from the subject. A method of the invention may comprise measuring the amount of phosphorylation at one, two, three, or all four of residues S428_ofXKB l,_S485-o£ AMPKα, Sl 08~of AMPKβ, or-

Sl 16 of eNOS III, the phosphorylation at other residues of these proteins that are associated with activation of the proteins, and/or the phosphorylation state of one or more (e.g., at least about 5, 10, 25, 50, 75 or 100) additional members of the AMPK or related pathways. AMPK pathway members include those described in WO/2004/050898.

Another aspect of the invention is a kit for determining if a subject having MLL is likely to be responsive to treatment with an inhibitor of a member of the AMPK signaling pathway, comprising reagents for measuring the amount of phosphorylation at one or more (e.g., one, two, three or all four) of residues S428 of LKBl, S485 of AMPKα, S108 of AMPKβ, or Sl 16 of eNOS III and, optionally, for measuring the amount of phosphorylation at one or more (e.g., at least about 5, 10, 25, 50, 75 or 100) additional members of the AMPK signaling pathway. The components of the kit may, optionally, be packaged in one or more containers.

Another aspect of the invention is a method comprising obtaining a tissue sample; obtaining data regarding the levels of phosphorylation of one or more of residues S428 of LKB 1 , S485 of AMPKα, S 108 of AMPKβ, or S 1 16 of eNOS III in the sample; and providing a report of those phosphorylation levels. The report may provide a comparison of the phosphorylation levels to baseline values.

A "subject," as used herein, includes any animal that has MLL. Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included. One suitable subject is the mouse model for MLL that is described in Example IV.

Sources of suitable "samples" will be evident to a skilled worker. Suitable samples comprise tissues or cells from a subject which contain one or more of the noted biomarkers, e.g., a raw PBMC isolate, or a PMBC that has been isolated by FACS, magnetic bead sorting, or a comparable technology. A "baseline value," as used herein, refers to the level of phosphorylation at a given amino acid residue of a protein (e.g., on the amino acid side chain) in a subject that is not responsive to an inhibitor of a phosphoprotein member of the noted AMPK pathway. An increase in the amount of phosphorylation of a protein (e.g., an increase in the total amount per cell of a phosphoprotein isoform of interest) can reflect an increased frequency of phosphorylation at the amino acid residue. In general, the total amount of protein that is phosphorylated at the noted amino acid residue is measured, per sample or per cell in the sample.

In one embodiment, the baseline value is determined by preparing positive and negative reference standards derived from tissue culture cells. To generate a "negative" reference standard, one can first process cells obtained from a suitable sample from a subject (or a pool of subjects) that is known to be non-responsive to an inhibitor of an AMPK pathway member. Protein extracts can be prepared from the sample and the level of phosphorylation (or range of values) at the phospho-endpoints of interest determined as described herein. The median value of such samples can serve as a negative reference standard. In one embodiment, the negative reference standard is a population average of samples from a pool of subjects having B-ALL.

To generate a "positive" reference standard, one can process cells from a comparable sample from a subject (or a pool of subjects) that is known to be responsive to responsive to an inhibitor of an AMPK pathway member. Protein extracts can be prepared from the sample and the level of phosphorylation (or range of values) at the phospho-endpoints of interest determined as described herein. The median value of such samples can serve as a positive reference standard.

In variations of the above method, the determination of the positive or negative standard may be based on published data, retrospective studies of patient samples, and other information that would be apparent to a person of ordinary skill implementing the methods of the invention. However, using such samples from subjects as a clinical diagnostic reference standard is generally not practical on a routine basis. Instead, it is preferable to generate negative and positive reference standards by using lysates from cells in culture, and establishing a cut-point value by a direct comparison of the cell culture lysates to a true positive (e.g. endpoint values derived from responsive subjects as described above) and true negative (e.g. endpoint values derived from non-responsive control subjects as described above). To accomplish this, one can first screen a variety of cells in culture, either primary cells or, preferably, cell lines.

The cells in culture can be propagated directly, under conventional conditions, so that the four proteins of the invention are not phosphorylated or are phosphorylated to a minimal degree; or they can be incubated under conventional conditions with a suitable mitogen that will globally activate signaling networks, such as pervanadate, or a growth factor, such as epidermal growth factor (EGF).

Protein extracts are then prepared from the various cell lines, which have been incubated under the various conditions, using conventional procedures; and the level of phosphorylation at the phospho-endpoints of interest determined as described herein, and compared directly to the true positive and true negative clinical samples as a bridging experiment. In this way, one can establish conditions such that particular cells, cultured under particular defined conditions (stimulated or not), express an amount of phosphorylation of the phosphoprotein isoforms of the invention that is directly comparable to those of a subject that is responsive to an inhibitor of an AMPK pathway member, or that is not responsive top such an agent. Utilizing the cut-point values derived from median values of known true clinical positives and negatives, and bridging these values to a cell line reference standard can then provide a " positive reference standard" or a " negative reference standard," respectively.

Alternatively, a baseline value can be the level of phosphorylation in a purified sample of the analyte (e.g., one or more of the phosphorylated protein isoforms of the invention) of known concentration. The baseline values may be selected using statistical tools that provide an appropriate confidence interval so that measured levels that are higher than the baseline value can be

accepted as being predictive of a positive response to therapy with an inhibitor of a phosphoprotein member of the AMPK pathway.

For each protein whose level of phosphorylation is determined, the value can be normalized, e.g., to the total protein in the cell; or to the amount of a constitutively expressed protein (from a housekeeping gene), such as actin; or the amount of a phosphoprotein may be compared to the amount of its non-phosphorylated counterpart.

Instead of using just a positive and negative reference standard, one can use a calibration curve that spans a range of values.

A "significantly elevated" level of phosphorylation (compared to a baseline value) is a level whose difference from the baseline value is statistically significant, using statistical methods that are appropriate and well-known in the art, generally with a probability value of less than five percent chance of the change being due to random variation. For example, the phosphorylation of the residues S428 of LKBl, S485 of AMPKoc, S 108 of AMPKβ, and/or Sl 16 of eNOS IH in a subject that is responsive to an inhibitor of a member of the AMPK pathway may range from about 0.25-fold to 10-fold higher (e.g. 5-fold higher), or more, than the level observed in a subject that is non-responsive.

The level of phosphorylation of a given amino acid residue can be measured qualitatively or quantitatively. The amount (quantity) of phosphorylation at a given residue may be higher than is observed at the same residue in a control sample (a baseline value). That is, it may be hyperphosphorylated. In addition to hyperphosphorylation as a detection threshold, the presence or absence of phosphorylation at the noted residues can also be utilized. Alternatively, a qualitative scale (such as a scale of 1 to 5) can be used.

Methods for measuring the level of phosphorylation at an amino acid residue are conventional and routine. In one embodiment, the measurement relies on the existence of sets of antibodies that are specific for either the non-phosphorylated or the phosphorylated forms of a particular amino acid residue of interest in the context of a protein of interest (such as phosphorylation of the noted residues of LKBl, AMPKα, AMPKβ, and/or eNOS III). Such antibodies are commercially available or can be generated routinely, using conventional procedures. In one embodiment, a synthetic peptide comprising an amino acid of interest from a protein of interest (either in the non-phosphorylated or phosphorylated form) is used as an antigen to prepare a suitable antibody. The antibody can be polyclonal or monoclonal.

Antibodies are selected and verified to detect only the phosphorylated version of the protein but not the non-phosphorylated version of the native or denatured protein, and vice-versa.

Such antibodies can be used in a variety of ways. For example, one can prepare whole cell lysates from patient samples and spot them in an array format onto a suitable substrate, such as nitrocellulose strips or glass slides. Preferably, the proteins in the samples are denatured before spotting. In general, the cells are spotted at serial dilutions, such as two-fold serial dilutions, to provide a wide dynamic range. Suitable controls, such as positive controls or controls for base line values, can be included. Each array is then probed with a suitable detectable antibody, as described above, to determine and/or to quantitate which amino acid residue(s) in the various proteins of interest are phosphorylated. Methods for immuno- quantitation are conventional. For a further discussion of the method of reverse phase protein lysate microarrays (RPMA), see, e.g., Nishizuka et al. (2003) Proc. Natl. Acad. Sci. 100, 14229- 14239.

Other suitable assays employing such antibodies to assess the level and/or degree of phosphorylation at a residue of interest include, e.g., Western blots, ELISA assays, immunoprecipitation, mass spectroscopy, and other conventional assays. Suitable methods include those that can detect the phosphoprotein in a very small sample {e.g. about 200 cells). Alternatively, methods can be used that are suitable for a large_sampleLsize_(e.g._about 20,000- 25,000 cells). Assays to measure the presence and/or amount of phosphorylated residues can be readily adapted to high throughput formats, e.g. using robotics.

One aspect of the invention is a method for treating a subject having MLL, comprising (1) measuring the amount of phosphorylation at residues S428 of LKB l, S485 of AMPKα, S 108 of AMPKβ, and/or Sl 16 of eNOS III in a sample from the subject and, if the levels of phosphorylation compared to a baseline value suggest that the subject is likely to be responsive to an inhibitor of a phosphoprotein member of the AMPK pathway, (2) administering an effective amount of an inhibitor of a member of the AMPK pathway to the subject.

Suitable inhibitory agents will be evident to a skilled worker. These include, e.g., the AMPK inhibitors, Dorsomorphin (6-[4-(2-Piperidin-l-yl-ethoxy)-phenyl)]-3-pyridin- 4-yl-pyrrazolo[l,5-a]-pyrimidine; Indirubin-3'-oxime or Indirubin-3'-monoxime (3-[l,3- Dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-l,3-dihydro-2H- indol-2-one); Iodotubercidin or 5-Iodotubercidin (4-Amino-5-iodo-7-(beta-D-ribofuranosyl) pyrrolo[2,3-d]-pyrimidine); or H-89

dihydrochloride or H89 (N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide . 2HC1); and the eNOS inhibitors, L-N5-(l-Iminoethyl)ornithine.2HCl; 1400W; Aminoguanidine,

Hemisulfate; l-Amino-2-hydroxyguanidine,/?-Toluenesulfonate; Bromocriptine Mesylate; Chlorpromazine, Hydrochloride; Dexamethasone; N ^G ,N ^G -Dimethyl-L-arginine,

Dihydrochloride; N ^G ,N° -Dimethyl-L-arginine, Dihydrochloride; Diphenyleneiodonium

Chloride; 2-Ethyl-2-thiopseudourea, Hydrobromide; or Haloperidol.

Methods for administering such inhibitors to a subject are conventional and well-known in the art. An "effective" amount of an inhibitor of the invention is an amount that is sufficient to elicit a measurable amount of a therapeutic activity. A subject that is "responsive" to an inhibitor of the invention exhibits a measurable decrease in one or more symptoms of MLL. In one embodiment of the invention, the progress of the disease is halted or even reversed.

Another aspect of the invention is a method for monitoring the effectiveness of a treatment of MLL. It is expected that level of phosphorylation at the residues discussed herein will remain at approximately the same level of decreased phosphorylation if the subject remains responsive to the therapy. However, if the subject begins to develop resistance to thejreatment, the levels of phosphorylation will increase. Such an increase would suggest that the AMPK inhibitor treatment should be halted. A method of the invention can be_used_to monitor- the- response to therapy by assessing the decrease in phosphorylation upon initial treatment, and monitoring patient AMPK pathway member baseline phosphorylation during treatment to assess if any resistance (e.g. increase in phosphorylation of any member of the AMPK pathway) occurs.

Another aspect of the invention is a kit useful for any of the methods disclosed herein. For example, the kit can be useful for predicting the response of a subject having MLL to a therapeutic method of the invention, comprising reagents for measuring the amount of phosphorylation at residues S428 of LKBl, S485 of AMPKα, S108 of AMPKβ, or Sl 16 of eNOS HI. The reagents can comprise, e.g., antibodies that are specific for particular unphosphorylated and/or phosphorylated isoforms of each of the three biomarkers that are identified herein. Furthermore, the kit may comprise reagents or devices for preparing a sample (e.g., for collecting a tissue and/or excising a sample from the tissue); for spotting test samples on a suitable surface, such as nitrocellulose strips; for performing immuno-quantitation (e.g., labeled antibodies, or reagents for labeling antibodies); instructions for performing a method of

the invention; etc. The components of the kit may, optionally, be packaged in one or more containers. Reagents for measuring phosphorylation at other residues of the noted proteins, or for measuring phosphorylation of other members of the AMPK pathway, can also be included.

Optionally, a kit of the invention comprises suitable buffers; one or more containers or packaging material; and/or a label indicating a use for the kit. The reagents of the kit may be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids. The reagents may also be in single use form, e.g., in a form for performing a single assay for one or more of the phosphorylated isoforms of the invention.

Suitable controls for assays of the invention will be evident to the skilled worker. For example, to provide for quality control, each set of proteins tested (e.g. in the form of a protein micro-array) may contain antigen controls, cell lysate controls, and/or a reference lysate. Each patient analyte sample can be normalized to total protein and quantitated in units relative to the reference "printed" on the same array. Each reference and control lysate can be printed in the same dilution series as patient samples and be immunostained at the same time, with identical reagents as the patient samples. All samples can be printed in duplicate or other multiples in, e.g., 4-point dilution curves.

To provide for quality assurance, samples can be processed and analyzed in real time, e.g. as they are received at a suitable processing facility that meets applicable regulatory standards. Samples may consist of Cytolyte preserved samples, or processed in any of a number of molecular fixatives that preserve important molecular information. A test set with matched frozen samples can verify the adequacy of specimen preservation. Techniques can be carried out at room temperature. Samples may be obtained by core needle biopsy.

Following the determination of the level of phosphorylation of a marker protein by a method as discussed herein, the values can be reported, e.g. in the form of a panel or suite of values, to physicians to improve therapy decisions for their patients. With such a report, subjects with MLL may be stratified at a molecular level, according to whether therapy with an inhibitor of a member of the AMPK pathway is likely to be effective. Some suitable systems for reporting the data are described in co-pending provisional application ser. no. 60/907,288, filed March 27, 2007. In the foregoing and in the following examples, all temperatures are set forth in uncorrected degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.

EXAMPLES

Example I - Materials and Methods

Patient studies: Reverse Phase Protein Arrays (RPPA) were employed to profile the working state of cellular STPs. The phosphorylation states of 92 key signalling proteins were analyzed from pediatric B-ALL or MLL specimens collected at disease presentation prior to treatment. Within this patient cohort, we compared 8 MLL patients (having the rearranged chromosomal abnormality associated with MLL) vs 39 patients having B-ALL (without known genomic aberrancies), to identify differentially activated phosphoproteins.

Pharmacological studies: Based on the results of the RPPA analysis, we then tested the effects of a specific AMPK inhibitor, (6-[4-(2-Piperidin-l-yl-ethoxy)-phenyl)]-3-pyridin-4-yl- pyrrazolo[l ,5-a]-pyrimidine) (6μM, 24hours), on the survival of two human cell lines: (1)

RS4;11, a human B-ALL cell line carrying the t(4;l l) MLL translocation; and (2) REH, a human B-ALL cell line carrying the t(12;21) translocation.

As a specificity control, we also used the Erk 1/2 inhibitor, l,4-diamino-2,3-dicyano-l,4- bis[2 aminophenylthio] butadiene) (5OuM, 24hours). We tested the apoptotic rate by Annexin V-PI cytofluorimetric assay.

Example II - Results of the patient study

Activation of the AMPK pathway is significantly elevated in patients with MLL compared to B-ALL patients (Figs. 3A, 3B and 3C). This pathway, through the phosphorylation of LKBl (S428) (Wilcoxon test, p=0.0157) and AMPK α (S485) (p=0.0019) and β (S108) (p=0.0006), leads to the activation of eNOS III (Sl 16) (p=0.0081). The activation of eNOS III leads to Nitric Oxide (NO) production. NO in undifferentiated tumors (such as MLL leukemias) is able to activate Bcl-2 (S70 p=0.0334 and T56 p=0.0122), and thus to inhibit the apoptotic process. Each of these endpoints lies in a common, linked pathway (sometimes referred to herein as the "AMPK pathway"), which indicates a pathway dergangement specific for MLL, and suggests individual therapies or combinations of therapies that can be used to treat MLL.

I l

Example III - Results of the pharmacological study

To show the functional significance of AMPK pathway activation, we treated RS4;11 and REH cells with an AMPK inhibitor (6-[4-(2-Piperidin-l-yl-ethoxy)-phenyl)]-3-pyridin-4-yl- pyrrazolo[l,5-a]-pyrimidine, 6μM, 24hours). After treatment, we observed that treated RS4;1 1 cells undergo significantly more apoptosis than REH cells (p<0.001): Cell viability after AMPK inhibition: 95% REH 36% RS4;1 1

As a specificity control, we also treated the cells with an Erkl/2 inhibitor (1,4-diamino- 2,3-dicyano-l,4-bis[2 aminophenylthio] butadiene, 50μM, 24hours, which is not differentially activated between MLL rearranged and not-rearranged patients. After treatment, RS4;11 and REH cells apoptotic rates are comparable: Cell viability after Erkl/2 inhibition: 94% REH 94% RS4;1 1 The data showing the specificity of therapeutic efficacy of drugs that specifically target the AMPK pathway are shown in Figures 4 and 5. FACS analysis of MLL (RS 4;11) and B-ALL cell lines (REH) show increased apoptosis only for the MLL phenotype when AMPK inhibitor is given. Moreover, use of other kinase inhibitors (e.g., an Erkl/2 inhibitor), unrelated to the MLL defect, have no discernable effect on MLL cell death.

In summary, the MLL leukemia patients (having the rearranged chromosomal abnormality) exhibit a statistically significant activation (phosphorylation) of the AMPK pathway compared to patients with B-ALL (without translocation). Without wishing to be bound by any particular mechanism, it is suggested that the activation of the AMPK pathway contributes to the survival of MLL leukemia cells, since it leads to Bcl-2 phosphorylation. Indeed, we found, surprisingly, that specific inhibition of AMPK induces death in MLL rearranged cancer cells, providing functional evidence for a causal underpinning for the pathway activation. The data suggest that AMPK inhibitors will be effective drugs for the treatment of MLL leukemia patients.

The ability to measure the indicated signalling pathways (as well as the functional activation of nearly 100 other signaling proteins at once) from a clinical sample can be used to identify chemical inhibitor therapeutics that can be used at the bedside, and thus can play an important role in improving treatment outcome through tailored therapy.

Example IV - Studies in an animal model

To show the functional significance of inhibiting the AMPK pathway activation in vivo, we employ a mouse model for human MLL. This preclinical mouse model comprises an Mll-Enl fusion protein which resulted from a Cre-IoxP-mediated interchromosomal translocation (Cano et al. (2008) MoI Cancer Ther l_, 730-5). The mice are treated with an AMPK inhibitor (6-[4-(2- Piperidin-l-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[l,5 -a]-pyrimidine,) 6μM (IC50) continuously for 2 weeks, and the effects of the compound are compared with and Erkl/2 inhibitor (l,4-diamino-2,3-dicyano-l,4-bis[2 aminophenylthio] butadiene, 50μM (IC50). In peripheral blood samples procured every 2 days from each mouse, we observe a statistically significant increase in apoptosis in the mice treated with the AMPK inhibitor compared to those treated with the ERX kinase inhibitor. Moreover, functional significance of the increase in cell death is seen by a dramatic reduction in the number of myeloid cells and a return to the normal lymphoid cell constituents.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make changes and modifications of the invention to adapt it to various usage and conditions and to utilize the present invention to its fullest extent. The preceding preferred specific embodiments are to be construed as merely illustrative, and not limiting of the scope of the invention in any way whatsoever. The entire disclosure of all applications, patents, and publications cited above and in the figures are hereby incorporated in their entirety by reference.