WO2007038406A1

WO2007038406A1 - Bax expression predicts hematologic disease response to treatment

Info

Publication number: WO2007038406A1
Application number: PCT/US2006/037210
Authority: WO
Inventors: Christopher Ramin Cogle; William Stratford May
Original assignee: University Of Florida Research Foundation, Inc.
Priority date: 2005-09-26
Filing date: 2006-09-26
Publication date: 2007-04-05
Also published as: WO2007038579A1

Abstract

The invention provides methods of determining the response of a patient with a hematologic disease to a drug by analyzing blood or bone marrow samples for levels of p21 Bax or p18 Bax or both p21 and p18 Bax.

Description

Bax Expression Predicts Hematologic Disease Response to Treatment

Cross Reference to Related Application

This application claims the benefit of priority U.S. Provisional Patent Application Serial Number 60/720,507, filed September 26, 2005, which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

Bax protein is essential for initiating cell death (Cao et al, Cleavage of Bax to pi 8 Bax accelerates stress-induced apoptosis, and a cathepsin-like protease may rapidly degrade pl8 Bax. Blood. 2003 Oct 1; 102(7): 2605-14). Following chemotherapy- induced stress, cytosolic Bax fully integrates into the outer mitochondrial membrane where it oligomerizes and forms death pores that facilitate the release of cytochrome c and other pro-apoptotic activators (Wood et al, Cleavage of Bax enhances its cell death function. Exp Cell Res. 2000 May 1; 256(2): 375-82; Gao et al, N-terminal cleavage of Bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2- independent cytochrome C release and apoptotic cell death. J Cell Biochem. 2000 Sep 18; 80(1): 53-72). A modified Bax protein (pl8 or truncated Bax) potently accelerates cell death (Cao et al). Moreover, decreased production of modified Bax has been associated with malignant progression. Certain hematologic diseases with higher or lower Bax expression may indicate the tendency or resistance for that cell to undergo chemotherapy-induced apoptosis.

Bax is cleaved by calpain at aspartate 33 (Asp33) to yield pl8 Bax during stress- induced apoptosis. An ecdysone-inducible expression system has been used to assess the role of pi 8 Bax in apoptosis. Similar levels of wild-type (WT) and noncleavable Asp33Ala (Asp~>Ala) Bax are induced in 293 cells while expression of N-terminal- deleted pi 8 (Deltal-33) Bax remains low (20% of full-length p21 Bax) due to a reduced half-life (2 hours versus 12 hours for p21 Bax) resulting from increased sensitivity to cathepsin-like proteolytic degradation. Expression of pl8 Bax is enhanced to levels comparable to p21 Bax when induction is carried out in the presence of cathepsin inhibitors, Z-Phe-Gly-NHO-Bz or N-Acetyl-Leu-Leu-Met-CHO. Compared with WT Bax ("p21 Bax"), expression of similar levels of pi 8 Bax and, surprisingly, Asp33Ala Bax more potently induces apoptosis as indicated by increased cytochrome c release, caspase- 9/-3 activation, and DNA fragmentation, potentially due to their increased homo- oligomerization in mitochondrial membranes. Studies in A-549, U-937, K-562, and HL- 60 cells confirm that inhibition of Bax cleavage results in 25% to 35% reduction of drug- induced apoptosis, while inhibition of pi 8 Bax degradation enhances apoptosis by 25% to 40%. Results indicate that although cleavage to pi 8 Bax is not required for Bax to initiate apoptosis, pi 8 Bax potently accelerates the apoptotic process. SUMMARY OF THE INVENTION

One embodiment of the invention provides a method of determining response of a patient with a hematologic disease to a drug. The method comprises contacting a blood sample or a bone marrow sample from the patient with the drug and analyzing the sample for levels of p21 Bax or pi 8 Bax or both p21 and pi 8 Bax. An increase in levels of p21 Bax or pi 8 Bax or both p21 Bax and pi 8 Bax as compared to the levels prior to treatment or to a control level indicates a positive response of the patient to the drug. The levels of p21 Bax and pi 8 Bax can be determined using antibodies specific for pi 8 Bax, antibodies specific for p21 Bax, antibodies specific for pl8 Bax and p21 Bax, or combinations thereof. The hematologic disease can be acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, extramedullar/ plasmacytoma, solitary myeloma, indolent myeloma, myelodysplastic syndrome, a myeloproliferative disorder, polycythemia vera, essential thrombocythemia, or idiopathic myelofibrosis. The drug can be daunorubicin, ATRA (all-trans retinoic acid), chlorambucil, cyclophosphamide, melphalan, doxorubicin, idarubicin, mitoxantrone, methotrexate, fludarabine, cytarabine, etoposide, topotecan, prednisone, dexamethasone, vincristine, vinblastine or combinations thereof.

Another embodiment of the invention provides a method of determining the response of a patient with a hematologic disease to treatment. The method comprises obtaining a blood sample or a bone marrow sample from the patient and analyzing the sample for levels of pi 8 Bax. An increase in levels of pi 8 Bax as compared to a control sample indicates a positive response of the patient to treatment.

Yet another embodiment of the invention provides a method of determining if a drug is effective against a hematologic disease. The method comprises contacting a blood sample or a bone marrow sample from a hematologic disease patient with the drug and analyzing the sample for levels of p21 Bax or pi 8 Bax or both p21 and pi 8 Bax. An increase in levels of p21 Bax or pl8 Bax or both p21 Bax and pl8 Bax indicates the drug is effective against a hematologic disease. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Spectrin degradation in leukemia cells exposed to chemotherapy. Lane 1 represents control lane. Lane 2 contains cell lysate proteins from cells exposed to just cytarabine pre-treatment. Lane 3 contains cell lysate proteins from cells exposed to just daunorubicin pre-treatment. Lane 4 contains cell lysate proteins from cells exposed to both cytarabine and daunorubicin. Spectrin degradation as a result of apoptosis is detected using antibodies directed against degradation products. This sample is from UPN 5 showing marked increased apoptosis when cells are exposed to daunorubicin.

Figure 2. Bax expression in leukemia cells exposed to chemotherapy. Lane 1 represents control lane. Lane 2 contains cell lysate proteins from cells exposed to just cytarabine pre-treatment. Lane 3 contains cell lysate proteins from cells exposed to just daunorubicin pre-treatment. Lane 4 contains cell lysate proteins from cells exposed to both cytarabine and daunorubicin. This sample is from UPN 5. Cell lysates after exposure to daunorubicin (lanes 3 and 4) demonstrate increased truncated Bax (pi 8). DETAILED DESCRIPTION OF THE INVENTION

The evaluation of Bax expression in patients with hematologic diseases enables prediction of disease response to therapy. For each type of hematologic disease, response to therapy can be quite heterogeneous. Some patients do better than others, largely for reasons unknown. Prognostic information identifying patients with good or poor predicted disease response to chemotherapy and low or high risk of disease relapse would be of great benefit.

One possible explanation of the heterogeneity in response to treatment among patients is the variability of diseased cells to undergo programmed cell death - apoptosis.

Apoptosis via the intrinsic cell death pathway is characterized by mitochondrial dysfunction followed by release of caspase activators that destroy cell structure and function Increased levels of pi 8 Bax in cells from patients with hematologic diseases correlates with chemosensitivity and potentially, disease remission. Therefore, detection of pl8 Bax can be used in prognosis of a patient at the time of diagnosis.

A hematologic disease is a cancer of the blood or bone marrow, such as leukemia or lymphoma. Specific types of hematologic diseases include, for example, acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, extramedullary plasmacytoma, solitary myeloma, indolent myeloma, myelodysplastic syndrome, and myeloproliferative disorders such as, polycythemia vera, essential (or primary) thrombocythemia, and idiopathic myelofibrosis.

Any drugs that can potentially treat a hematologic disease can be used in the methods of the invention, e.g., daunorubicin, ATRA (all-trans retinoic acid), chlorambucil, cyclophosphamide, melphalan, doxorubicin, idarubicin, mitoxantrone, imethotrexate, fludarabine, cytarabine, etoposide, topotecan, prednisone and dexamethasone, vincristine and vinblastine. Additionally, any type of experimental drugs can be tested for efficacy in treating hematologic diseases. Prognostic Assays

Methods of the invention provide assays for determining the response of a patient with a hematologic disease to a drug treatment. A blood sample or a bone marrow sample from the patient can be contacted with the drug. The blood or bone marrow sample is then analyzed for the level of the p21 Bax or pl8 Bax or both p21 and pl8 Bax. An increase in levels of p21 Bax or pl8 Bax or both p21 Bax and pl8 Bax as compared to the levels prior to the treatment or to control levels indicates a positive response of the patient to the drug.

Additionally, methods of determining response of a patient with a hematologic disease to treatment are provided by the invention. The methods comprise obtaining a blood sample or a bone marrow sample from the patient and analyzing the sample for levels of pi 8 Bax an increase in levels of pi 8 Bax as compared to a control sample indicates a positive response of the patient to treatment.

The invention also provides methods of determining if a drug is effective against a hematologic disease. A blood sample or a bone marrow sample from a hematologic disease patient is contacted with one or more drugs. The blood or bone marrow samples are analyzed for levels of p21 Bax or pi 8 Bax or both p21 and pl8Bax. An increase in levels of p21 Bax or pi 8 Bax or both p21 Bax and pi 8 Bax indicates the drug is effective against a hematologic disease.

Any type of blood or bone marrow samples can be used in the instant invention. For example, human or animal blood samples can be used. Blood samples can include, e.g., plasma, serum, whole blood or peripheral blood mononuclear cells.

An antibody, aptamer, or nanoparticle can be used to detect p21 Bax and/or pl8 Bax. For example, an antibody that binds to both pl8 and to p21 Bax can be used. The two forms can then be differentiated by size analysis. An antibody can be used that binds both pi 8 and p21 Bax along with an antibody that binds to one or more of the 33 N- terminal amino acids of p21 Bax (this antibody would not bind to pi 8 Bax since pi 8 Bax does not have the 33 N-terminal amino acids of p21 Bax). Alternatively, an antibody that binds p21 Bax exclusively and one that binds pi 8 Bax exclusively can be used. Antibodies to Bax are commercially available from, e.g., Santa Cruz Biotechnology, me, Santa Cruz, CA; Abeam, Cambridge, MA; Accurate Chemical, Westbury, NY; Axxora, San Diego, CA; Sigma Aldrich; Biodesign International, Saco, Maine; Biogenesis, Kinston NH; Biogenex, San Romon, CA; Southern Biotechnology Assoc, Birmingham AL, Bionostics, Devens, MA; Biosource, Camarillo, CA; Trevigen, Gaithersburg, MD.

Antibodies of the invention are antibody molecules that specifically and stably bind to pi 8 Bax or p21 Bax or both. An antibody of the invention can be a polyclonal antibody, a monoclonal antibody, a single chain antibody (scFv), or a fragment of an antibody. Fragments of antibodies are a portion of an intact antibody comprising the antigen binding site or variable region of an intact antibody, wherein the portion is free of the constant heavy chain domains of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')₂ and F_v fragments.

An antibody of the invention can be any antibody class, including for example, IgG, IgM, IgA, IgD and IgE. An antibody or fragment thereof binds to an epitope of a polypeptide of the invention. An antibody can be made in vivo in suitable laboratory animals or in vitro using recombinant DNA techniques. Means for preparing and characterizing antibodies are well know in the art. See, e.g., Dean, Methods MoI. Biol. 80:23-37 (1998); Dean, Methods MoI Biol. 32:361-79 (1994); Baileg, Methods MoI Biol. 32:381-88 (1994); Gullick, Methods MoI. Biol 32:389-99 (1994); Drenckhahn et al. Methods Cell. Biol. 37:7-56 (1993); Morrison, Ann. Rev. Immunol. 10:239-65 (1992); Wright et al. Crit. Rev. Immunol 12:125-68 (1992). For example, polyclonal antibodies can be produced by administering p 18 Bax, p21 Bax, or a fragment thereof to an animal, such as a human or other primate, mouse, rat, rabbit, guinea pig, goat, pig, dog, cow, sheep, donkey, or horse. Serum from the immunized animal is collected and the antibodies are purified from the plasma by, for example, precipitation with ammonium sulfate, followed by chromatography, such as affinity chromatography. Techniques for producing and processing polyclonal antibodies are known in the art.

"Specifically binds" or "specific for" means that the polypeptide recognizes and binds to an antibody of the invention with greater affinity than to other, non-specific molecules. For example, an antibody raised against an antigen (e.g., a polypeptide) to which it binds more efficiently than to a non-specific protein can be described as specifically binding to the antigen. Specific binding can be tested using, for example, an enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), or a western blot assay using methodology well known in the art.

Additionally, monoclonal antibodies directed against epitopes present on pi 8 Bax or p21 Bax can also be readily produced. For example, normal B cells from a mammal, such as a mouse, which was immunized with a Bax polypeptide can be fused with, for example, HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomas producing Bax-specific antibodies can be identified using RIA or ELISA and isolated by cloning in semi-solid agar or by limiting dilution. Clones producing Bax-specific antibodies are isolated by another round of screening. Monoclonal antibodies can be screened for specificity using standard techniques, for example, by binding a Bax polypeptide to a microtiter plate and measuring binding of the monoclonal antibody by an ELISA assay. Techniques for producing and processing monoclonal antibodies are known in the art. See e.g., Kohler & Milstein, Nature, 256:495 (1975). Particular isotypes of a monoclonal antibody can be prepared directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of a different isotype by using a sib selection technique to isolate class-switch variants. See Steplewski et al, P.N.A.S. U.S.A. 82:8653 1985; Spria et al, J. Immunolog. Meth. 74:307, 1984. Monoclonal antibodies of the invention can also be recombinant monoclonal antibodies. See, e.g., U.S. Patent No. 4,474,893; U.S. Patent No. 4,816,567. Antibodies of the invention can also be chemically constructed. See, e.g., U.S. Patent No. 4,676,980.

Antibodies of the invention can be chimeric {see, e.g., U.S. Patent No. 5,482,856), humanized (see, e.g., Jones et al, Nature 321:522 (1986); Reichmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992)), or human antibodies. Human antibodies can be made by, for example, direct immortilization, phage display, transgenic mice, or a Trimera methodology, see e.g., Reisener et al, Trends Biotechnol. 16:242-246 (1998).

Antibodies that specifically bind Bax antigens (e.g., Bax polypeptides), are particularly useful for detecting the presence of Bax in a sample, such as a serum, blood, or bone marrow sample from an animal such as a human. An immunoassay for Bax can utilize one antibody or several antibodies. An immunoassay for Bax can use, for example, a monoclonal antibody directed towards a Bax epitope, a combination of monoclonal antibodies directed towards epitopes of one Bax polypeptide, monoclonal antibodies directed towards epitopes of different Bax polypeptides, polyclonal antibodies directed towards the same Bax antigen, polyclonal antibodies directed towards different Bax antigens, or a combination of monoclonal and polyclonal antibodies. Immunoassay protocols can be based upon, for example, competition, direct reaction, or sandwich type assays using, for example, labeled antibody. Antibodies of the invention can be labeled with any type of label known in the art, including, for example, fluorescent, chemiluminescent, radioactive, enzyme, colloidal metal, radioisotope and bioluminescent labels.

Antibodies of the invention or fragments thereof can be bound to a support and used to detect the presence of Bax. Supports include, for example, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magletite.

Antibodies of the invention can further be used to isolate Bax by immunoaffϊnity columns. The antibodies can be affixed to a solid support by, for example, adsorbtion or by covalent linkage so that the antibodies retain their immunoselective activity. Optionally, spacer groups can be included so that the antigen binding site of the antibody remains accessible. The immobilized antibodies can then be used to bind Bax antigens from a sample, such as a biological sample including, e.g., blood or bone marrow. The bound Bax is recovered from the column matrix by, for example, a change in pH. Methods of Detection

The methods of the invention can be used to detect pi 8 Bax and/or p21 Bax in a test sample, such as blood or bone marrow. The test sample can be untreated, precipitated, fractionated, separated, diluted, concentrated, or purified.

The methods can comprise contacting antibodies specific for pi 8 Bax and/or p21 Bax with a test sample under conditions that allow a polypeptide/antibody complex, i.e., an immunocomplex, to form. That is, antibodies bind to pi 8 Bax and/or p21 Bax located in the sample. One of skill in the art is familiar with assays and conditions that are used to detect antibody/polypeptide complex binding. The formation of a complex between Bax and antibodies in the sample is detected. The amount of antibody-antigen complexes can be determined by methodology known in the art.

In one embodiment of the invention, the polypeptide/antibody complex is detected when an indicator reagent, such as an enzyme conjugate, which is bound to the antibody, catalyzes a detectable reaction. Optionally, an indicator reagent comprising a signal generating compound can be applied to the polypeptide/antibody complex under conditions that allow formation of a polypeptide/antibody/indicator complex. The polypeptide/antibody/indicator complex is detected. Optionally, the polypeptide or antibody can be labeled with an indicator reagent prior to the formation of a polypeptide/antibody complex. The method can optionally comprise a positive or negative control.

In one embodiment of the invention, antibodies specific for Bax are attached to a solid phase or substrate. A test sample potentially comprising Bax is added to the substrate. Antibodies that specifically bind pi 8 Bax and/or p21 Bax are added. The antibodies can be the same antibodies used on the solid phase or can be from a different source or species and can be linked to an indicator reagent, such as an enzyme conjugate. Wash steps can be performed prior to each addition. A chromophore or enzyme substrate is added and color is allowed to develop. The color reaction is stopped and the color can be quantified using, for example, a spectrophotometer.

Assays of the invention include, but are not limited to those based on competition, direct reaction or sandwich-type assays, including, but not limited to enzyme linked immunosorbent assay (ELISA), western blot, IFA, radioimmunoassay (RIA), hemagglutination (HA), fluorescence polarization immunoassay (FPIA), and microtiter plate assays (any assay done in one or more wells of a microtiter plate).

Assays can use solid phases or substrates or can be performed by immunoprecipitation or any other methods that do not utilize solid phases. Where a solid phase or substrate is used, an antibody is directly or indirectly attached to a solid support or a substrate such as a microtiter well, magnetic bead, non-magnetic bead, column, matrix, membrane, fibrous mat composed of synthetic or natural fibers (e.g., glass or cellulose-based materials or thermoplastic polymers, such as, polyethylene, polypropylene, or polyester), sintered structure composed of particulate materials (e.g., glass or various thermoplastic polymers), or cast membrane film composed of nitrocellulose, nylon, polysulfone or the like (generally synthetic in nature). All of these substrate materials can be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics. Suitable methods for immobilizing antibodies on solid phases include ionic, hydrophobic, covalent interactions and the like.

The formation of a polypeptide/antibody complex or a polypeptide/antibody/indicator complex can be detected by radiometric, colormetric, fluorometric, size-separation, or precipitation methods. Optionally, detection of a polypeptide/antibody complex is by the addition of a secondary antibody that is coupled to an indicator reagent comprising a signal generating compound. Indicator reagents comprising signal generating compounds (labels) associated with a polypeptide/antibody complex can be detected using the methods described above and include chromogenic agents, catalysts such as enzyme conjugates fluorescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums, ruthenium, and luminol, radioactive elements, direct visual labels, as well as cofactors, inhibitors, magnetic particles, and the like. Examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances. EXAMPLES

The objectives of this example were to correlate pi 8 Bax in cancer cells with clinical outcome. The strategy of the study was to evaluate circulating blood cells in patients with cancer, in order to provide better prognostic information and identify molecular mechanisms to increase chemotherapy sensitivity.

To determine if Bax predicts for disease response to treatment, five patients referred to our leukemia program for evaluation and treatment were enrolled (Table 1).

Peripheral blood mononuclear cells from the patients were isolated by Ficoll separation. Cells were then treated with cytarabine, daunorubicin, or a combination of cytarabine and daunorubicin. Cytarabine treated cells were exposed to a 20 micromolar for 24 hours. For daunorubicin treatment, cells were exposed to a concentration of 0.5 micrograms per milliliter for 24 hours. Cells were then lysed. Proteins were analyzed by Western blot. The blots were probed with anti-human spectrin degradation products, Bax (p21), and truncated Bax (pi 8).

The 120 kDa spectrin degradation production reproducibly demonstrated a sharp band correlating with degree of apoptosis (Figure 1). Probing for p21 Bax and pi 8 Bax demonstrated different quantities of protein depending on chemotherapy preparation (Figure 2). In particular, Bax expression demonstrated no increase in patients that did not achieve a remission of their disease (Table 1).

These results show that the level of Bax (both p21 and pi 8) correlates with treatment response. Quite significantly, the results of treatment sensitivity are available at the time of diagnosis. Thus, measurement of Bax expression could help predict response to treatment. In addition, our results highlight the importance of Bax in hematologic disease. Increasing Bax expression may augment treatment-induced apoptosis. Moreover, administering inhibitors of Bax degradation (such as targeting calpain inhibitors) would increase the level of Bax, thus make the cell more susceptible to therapy-induced apoptosis. Ultimately this would lead to better treatment response in patients.

Claims

CLAIMSWe claim:

1. A method of determining response of a patient with a hematologic disease to a drug comprising:

(a) contacting a blood sample or a bone marrow sample from the patient with the drug;

(b) analyzing the sample for levels of p21 Bax or pl8 Bax or both p21 and pi 8 Bax; wherein an increase in levels of p21 Bax or pi 8 Bax or both p21 Bax and pi 8 Bax as compared to the levels prior to treatment or to a control level indicates a positive response of the patient to the drug.

2. The method of claim 1, wherein the levels of p21 Bax and pl8 Bax are determined using antibodies specific for pi 8 Bax, antibodies specific for p21 Bax, antibodies specific for pi 8 Bax and p21 Bax, or combinations thereof.

3. The method of claim 1, wherein the hematologic disease is acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, extramedullary plasmacytoma, solitary myeloma, indolent myeloma, myelodysplastic syndrome, a myeloproliferative disorder, polycythemia vera, essential thrombocythemia, or idiopathic myelofibrosis.

4. The method of claim 1, wherein the drug is daunorubicin, ATRA (all-trans retinoic acid), chlorambucil, cyclophosphamide, melphalan, doxorubicin, idarubicin, mitoxantrone, imethotrexate, fludarabine, cytarabine, etoposide, topotecan, prednisone, dexamethasone, vincristine, vinblastine or combinations thereof.

5. A method of determining response of a patient with a hematologic disease to treatment comprising:

(a) obtaining a blood sample or a bone marrow sample from the patient;

(b) analyzing the sample for levels of pi 8 Bax; wherein an increase in levels of pi 8 Bax as compared to a control sample indicates a positive response of the patient to treatment.

6. The method of claim 5, wherein the levels of pl8 Bax are determined using antibodies specific for pi 8 Bax.

7. The method of claim 5, wherein the hematologic disease is acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, extramedullary plasmacytoma, solitary myeloma, indolent myeloma, myelodysplastic syndrome, a myeloproliferative disorder, polycythemia vera, essential thrombocythemia, or idiopathic myelofibrosis.

8. The method of claim 1, wherein the drug is daunorubicin, ATRA (all-trans retinoic acid), chlorambucil, cyclophosphamide, melphalan, doxorubicin, idarubicin, mitoxantrone, imethotrexate, fludarabine, cytarabine, etoposide, topotecan, prednisone, dexamethasone, vincristine, vinblastine or combinations thereof.

9. A method of determining if a drug is effective against a hematologic disease comprising:

(a) contacting a blood sample or a bone marrow sample from a hematologic disease patient with the drug;

(b) analyzing the sample for levels of p21 Bax or pi 8 Bax or both p21 and pi 8 Bax; wherein an increase in levels of p21 Bax or pi 8 Bax or both p21 Bax and pi 8 Bax indicates the drug is effective against a hematologic disease.

10. The method of claim 9, wherein the levels of p21 Bax and pl8 Bax are determined using antibodies specific for pi 8 Bax, antibodies specific for p21 Bax, antibodies specific for pi 8 Bax and p21 Bax, or combinations thereof.

11. The method of claim 9, wherein the hematologic disease is acute myelogenous leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, extramedullary plasmacytoma, solitary myeloma, indolent myeloma, myelodysplastic syndrome, a myeloproliferative disorder, polycythemia vera, essential thrombocythemia, or idiopathic myelofibrosis.

12. The method of claim 9, wherein the drug is daunorubicin, ATRA (all-trans retinoic acid), chlorambucil, cyclophosphamide, melphalan, doxorubicin, idarubicin, mitoxantrone, imethotrexate, fludarabine, cytarabine, etoposide, topotecan, prednisone, dexamethasone, vincristine, vinblastine or combinations thereof.