WO2000028813A9 - Treatment of myelogenous leukemia - Google Patents

Treatment of myelogenous leukemia

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Publication number
WO2000028813A9
WO2000028813A9 PCT/US1999/027392 US9927392W WO0028813A9 WO 2000028813 A9 WO2000028813 A9 WO 2000028813A9 US 9927392 W US9927392 W US 9927392W WO 0028813 A9 WO0028813 A9 WO 0028813A9
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WIPO (PCT)
Prior art keywords
abl
bcr
cells
temperature
patient
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PCT/US1999/027392
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French (fr)
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WO2000028813A1 (en
Inventor
Lyuba Varticovski
Suresh K Jain
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St Elizabeth S Medical Ct
Lyuba Varticovski
Suresh K Jain
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Application filed by St Elizabeth S Medical Ct, Lyuba Varticovski, Suresh K Jain filed Critical St Elizabeth S Medical Ct
Priority to AU20264/00A priority Critical patent/AU2026400A/en
Publication of WO2000028813A1 publication Critical patent/WO2000028813A1/en
Publication of WO2000028813A9 publication Critical patent/WO2000028813A9/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0081—Purging biological preparations of unwanted cells
    • C12N5/0093—Purging against cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms

Definitions

  • the present invention relates to method for treating myelogenous leukemia.
  • Leukemia is a malignant disease of the bone marrow and blood.
  • the common types of leukemia are divided into myelogenous leukemia and lymphocytic leukemia.
  • Lymphocytic leukemia involves uncontrolled growth of lymphocytes in the marrow, leading invariably to an increase in the concentration of lymphocytes in the blood.
  • Lymphocytes are cells that mediate the specificity of the immune response.
  • myeloid leukemia also called non- lymphocytic or granulocytic leukemia
  • myeloid leukemia also called non- lymphocytic or granulocytic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • CML chronic myelomonocytic leukemia
  • CCMOL chronic monocytic leukemia
  • MDS myelodysplastic syndrome
  • CML is characterized cytologically by the Philadelphia (Ph) chromosome, which results from the translocation of the Abelson (abl) oncogene on chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22.
  • the DNA from chromosome 9 contains most of the proto- oncogene designated c-abl.
  • the break in chromosome 22 occurs in the middle of the bcr gene.
  • the resulting Ph chromosome has the 5' section of bcr fused with most of c-abl.
  • the result of this translocation is the expression of a constitutively active cytoplasmic protein-tyrosine kinase activity, BCR/abl. (Clark, S.S., et al., (1989) Annu. Rev. Med., 40; 113- 122; Groffen, J., et al., (1984) Cell, 36; 93-99).
  • the BCR/abl oncogene most frequently encountered in human leukemias encodes frequently two proteins, p l85 (frequently referred to in the literature as p l90), with smaller BCR domain (amino acids 1-426) and p210, with a larger BCR domain (amino acids 1-902 or 926).
  • p 185 is associated with acute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML) and p210 is found in almost all cases of chronic myelogenous leukemia (CML).
  • BCR/abl Activated tyrosine kinase of abl is required for transformation of hematopoietic cells and triggers multiple signaling pathways.
  • the transforming capacity of BCR/abl is based in its enhanced tyrosine kinase activity.
  • BCR/abl expression in cytokine-dependent murine hematopoietic cell lines leads to growth factor independence (Daley GQ, et al, Proc Natl Acad Sci, USA 1988; 85: 9312-9316; Hariharan IK, et al., Oncogene 1988; 3: 387- 399) and protection of cells from apoptosis induced by irradiation or cytotoxic agents.
  • CML progenitor cells are growth factor independent in addition to resistant to apoptotic stimuli is currently under investigation.
  • Fever is a key feature of infection and inflammation, and is a common feature in autoimmune diseases.
  • In vitro hyperthermia has been shown to potentiate immune responses to IL- 1 , IL-2 and immune response to antigen including enhanced antigen processing.
  • lymphocytic leukemia is a disease of the immune system
  • hyperthermia has not been considered as a treatment option for CML.
  • a transplant is necessary because any treatment that removes all the BCR/abl tumor cells from the marrow also kills the stem cells which are known to express BCR/abl, leaving the patient with no ability to make new blood cells.
  • An allogeneic bone marrow transplant is the transfer of stem cells from a donor to the recipient CML patient and autologenous transplant is the transfer of stem cells from CML patient in remission, after treatment, to the same patient.
  • the present invention provides an ex ⁇ ivo method of reducing tumor cells in bone marrow or stem cells collected from a patient suffering from myelogenous leukemia, particularly chronic myelogenous leukemia (CML), prior to transplant into the patient.
  • the method comprises subjecting the bone marrow or stem cells to a temperature above 37°C for a period of time sufficient to inactivate tumor cells contained therein.
  • the temperature is raised within a range of about 38°C to about 42°C, preferably about 39°C, for a period time sufficient to deactivate tumor cells contained therein, preferably for about 2 to 72 hours, more preferably for about 12 to 24 hours and most preferably about 16 to 18 hours.
  • These methods preferably further comprise administering at least one anti-tumor cell agent, e.g., anti-CML agent, to the bone marrow or collected stem cells prior to, during, or after the heat treatment.
  • at least one anti-tumor cell agent e.g., anti-CML agent
  • stem cell preparations includes preparations containing stem cells from bone marrow or peripheral blood.
  • the present invention further provides a method for treating myelogenous leukemia, particularly CML, comprising subjecting a patient having the disease to whole-body hyperthermia (WBH), wherein the body temperature of the patient is raised to a temperature above 37°C.
  • WBH whole-body hyperthermia
  • the body temperature is raised within a range of about 39°C to about 42°C, preferably about 30°-40°C, for about 2 hours to about 24 hours, preferably about 16 to 18 hours.
  • the present invention also provides a method for treating myelogenous leukemia comprising subjecting a patient having CML to whole-body hyperthermia (WBH), and administering at least one anti-CML agent to the patient prior to, during, or after the patient has undergone whole body hyperthermia.
  • Anti-CML agents include conventional therapy of chemotherapy and biologic response modifiers.
  • the treatment can be repeated at periodic intervals, e.g., about 48 to 72 hours, preferably about every 48 hours, for a period of 7 to 10 days, preferably for a week.
  • the present invention also provides a method of screening compounds for treating chronic myelogenous leukemia comprising subjecting an animal having CML to whole-body hyperthermia (WBH), wherein the body temperature of the animal is raised above its normal body temperature, administering at least one compound or composition to be evaluated for anti-CML properties to the animal prior to, during, or after the animal has undergone whole body hyperthermia and determining the effectiveness of the compound by evaluating the effect of the treatment on the animal's CML cells.
  • WBH whole-body hyperthermia
  • materials produced by or associated with CML cells such as BCR/abl tyrosine kinase activity, tyrosine phosphorylated proteins, PI 3-kinase activity, or by other methods including but not limited to cell number, cell survival and apoptosis.
  • the present invention also provides a method of downregulating abl kinase activity of BCR/abl and v-abl transformed hematopoietic cells comprising subjecting the cells to temperatures above 37°C. This method results in reduction of tyrosine phosphorylated substrates in hematopoietic cells.
  • FIG. 1A shows parental BaF (lanes 1, 2), hematopoietic cells expressing wild type (wt) p210 BCR/abl, BaFp210 (lanes 3, 4), 32Dp210 (lanes 5,6) and FDCPp210 (lanes 7,8).
  • Figure IB shows the time course of BCR/abl tyrosine kinase downregulation at 39°C.
  • Figure 1C shows that temperature-induced downregulation of BCR/abl tyrosine kinase in CMb cells is potentially reversible.
  • Figure 2 shows the downregulation of BCR/abl tyrosine kinase at 39°C is not influenced by BCR/abl structural motifs and is evident only when expressed in hematopoietic cells.
  • Figure 2A is a graphic representation of wt and mutants of BCR/abl and Abl.
  • Figure 2B shows that BCR/abl structural domains do not affect the kinase activity at 39°C.
  • Figure 2C shows BCR/abl, Abl and PDGF-R kinase activities are not downregulated in NIH3T3 fibroblasts by increased temperature.
  • Figure 3 shows the effect of temperature on p210 BCR/abl and recombinant purified Abl kinase.
  • Figure 3 A shows a graph of kinase activity determined by the rate of 32 P incorporation into raytide substrate.
  • Figure 3B shows the effect of temperature on autophosphorylation of immunoprecipitated BCR/abl protein.
  • Figure 3C shows the effect of temperature on the kinase activity of purified recombinant Abl.
  • Figure 4 shows that expression or activity of protein tyrosine phosphatases in hematopoietic cell species is not altered by temperature shift.
  • Figure 4A shows expression of FTP- IB, SH-PTPl and SH-PTP2 protein tyrosine phosphatases in BaFp210 cells at 39°C.
  • Figure 4B shows is a graph showing total cellular protein tyrosine phosphatase activity is not altered in BaFp210 cells grown at 39°C.
  • Figure 5 shows that down-regulation of PI 3-kinase activity by temperature shift at 39°C does not correlate with proliferation of hematopoietic cells.
  • Figure 5A shows temperature-induction of a reversible downregulation of PI 3-kinase.
  • Figure 5B shows that downregulation of BCR/abl activity by temperature does not affect cell growth of the BaFp210 cell line or IL-3 independence.
  • the present inventors have surprisingly discovered that BCR/abl tyrosine kinase activity is dramatically reduced in hematopoietic cells maintained in culture at temperatures above normal body temperature.
  • the expression of BCR/abl is unaffected and tyrosine kinase activity can be recovered when cells are shifted back to 37°C.
  • the down regulation of kinase activity increases the number of tumor cells killed during this period, providing an ex vivo method to reduce tumor cells in bone marrow to be used in treating patients having CML or in stem cell preparations from peripheral blood.
  • the patient's own bone marrow or collected stem cells are subjected ex-vivo to a temperature above 37°C for a period of time sufficient to kill any residual tumor cells.
  • an anti-tumor cell agent e.g., anti-CML agent
  • the anti-CML agent may be added prior to, during or after hyperthermic treatment.
  • the bone marrow or stem cell preparation is subjected to the increased temperature for a period of time that is sufficient to kill the tumor cells, while minimizing the adverse effects on normal cells in the marrow or preparation.
  • a preferred range of time for treatment is from about 2 to 72 hours, more preferably about 10 to 24 hours, most preferably about 16 hours.
  • blood e.g., collected peripheral blood and blood products may be treated with the same ex vivo treatment to kill tumor cells prior to transfusion.
  • treatments can be used on the patient's own blood or blood products to eliminate tumor cells or a donor's blood or blood products.
  • the blood or blood product is subjected to a temperature above 37°C to kill tumor cells.
  • an anti-CML agent may be added to the blood or blood product, prior to, during or after hyperthermic treatment.
  • Anti-CML agents useful in the practice of the present invention include conventional chemotherapy and biologic response modifiers, e.g., interferon. See, Katarjian, H.P., et al., Blood, 82: 691-703, 1993.
  • Additional anti-CML agents include tyrosine kinase inhibitors that can be used to block BCR/abl tyrosine kinase activity.
  • Such inhibitors include, for example, tyrphostins, 2-phenylaminopyrimidine derivatives, pyrazolpyrimidines, banzopyranones and benzothiopyranones, herbimycin A, and glendamycin.
  • the body temperature of the patient is raised to a temperature in the range of about 38°C to about 42°C, preferably about 39°C, for about 2 hours to about 24 hours, preferably about 16 hours.
  • the treatment can be repeated at periodic intervals, e.g., 16 hours, every 2-3 days for a week, to eliminate the tumor cells.
  • Anti-CML agents can be administered to the patient prior to, during, or after the patient has undergone whole body hyperthermia.
  • hyperthermia is a treatment in which the body temperature is raised from 37°C up to 42°C.
  • Hyperthermia has the ability to selectively kill some heat sensitive malignancies.
  • hyperthermia can be combined with other treatments (for example, chemotherapy) to increase their effect.
  • chemotherapy for example, chemotherapy
  • Body temperature can be raised by a number of mechanisms which are known in the art.
  • techniques include paraffin wax baths, high temperature hydro therapy with immersion, hot warming blankets and insulation, forms of radiant energy and extracorporeal circulation.
  • One whole body hyperthermia device called the "Aquatherm" utilizes a radiant heat technology.
  • the radiant heat produced by the Aquatherm produces heat while air temperatures are elevated only minimally.
  • This device uses a water tight cylindrical coil through which heated water can be pumped while maintaining low pressure. Design features for this device include a counter- current flow pattern to maintain temperature homogeneity.
  • a humidification system maintains humidity within the chamber.
  • the Aquatherm controls radiant heat exchanges to supplement metabolic heat production while minimizing evaporative heat losses.
  • the subject to be treated lies on a stretcher which is mounted on rails to allow easy movement in and out of the device.
  • a non-conductive plastic netting is provided inside the heating chamber to prevent patient contact with the inner heat-radiating surface of the chamber.
  • An industry standard 40 W light is incorporated into the rear wall of the device to maximize patient observations during treatments.
  • Two thermal insulating plexiglass doors are provided at the head of the apparatus. These doors allow the patient's head to remain outside the heating chamber at all times; they also allow for patient observation.
  • a soft collar is incorporated into the door design which fits around the neck of the patient. This apparatus is disclosed in U.S. Pat. No. 5,713,941, the disclosure of which is herein incorporated by reference.
  • monitoring devices are attached to the patient. These monitors assist the hyperthermia team in ensuring patient comfort and safety. Monitoring includes body temperatures, heart rate, blood pressure, urine production, breathing rate, and state of consciousness.
  • An IN. is started for the purpose of administering fluids and medications. After the necessary pretreatment preparations are made, the patient's body, except for the head and neck, remains in the Aquatherm device for approximately 1.5 hours in order to achieve the desired temperature. At this time the patient is removed from the device and covered with blankets. While covered, the higher body temperature is maintained for the period of time specified in each treatment plan. During the heating and treatment phases, medications are administered for patient comfort. The patient will feel drowsy during the treatment but is able to talk with the hyperthermia team. At the conclusion of the treatment, the heat- retaining blankets are removed and body cooling begins. Monitoring of the patient continues until body temperature returns to the normal range. Generally, treatment duration from start to finish is less than 16 hours.
  • the present inventors demonstrate that tyrosine kinase activity of wild type BCR/abl is temperature sensitive in vitro, when expressed in hematopoietic cells. Tyrosine phosphorylation of cellular substrates and PI 3-kinase activity in BCR/abl and v-abl transformed hematopoietic cells are abrogated by overnight exposure to 39°C without change in protein expression. This effect is reversible, and phosphorylation of intracellular proteins is fully restored at 37°C within 4-12 hours.
  • the methods of the present invention are selective for abl kinase. Temperature-induced inhibition of Abl kinase activity in vivo in cell lines was detected only in hematopoietic cells and did not occur in NIH3T3 fibroblasts which express the same gene products. Activation of PDGF-R kinase was also not altered after prolonged incubation of NIH3T3 fibroblasts at 39°C. Downregulation of Abl kinase activity was not induced when expressed in NIH3T3 fibroblasts.
  • BCR/abl-induced genetic instability may account for secondary mutations which provide alternative mechanisms for cell survival and no longer require active BCR/abl.
  • low levels of BCR/abl tyrosine kinase activity may be sufficient to maintain proliferation of these cells whereas higher levels of kinase activity are necessary for protection against apoptosis.
  • a dose dependent hierarchy for BCR/abl induced biological effects in hematopoietic cells was previously proposed. ⁇ The increase in temperature may also have an independent effect on activation of other survival pathways which potentiate biological response to BCR/abl.
  • the present invention also relates to methods of screening compounds for use as anti-CML agents.
  • CML does not spontaneously occur in animals
  • animal models for studying CML are now known in the art. (See e.g., Daley, G.Q., et al., Science, 1990, 247:4944, 824-30; Daley, G.Q., et al., Proc. Natl. Acad. Sci., 1991 , 88:24, 11335-8; Ilaria, R.L., et al., Blood, 1995, 86: 10, 3897-904).
  • These animal models can be used to develop and test potential compounds for use as anti-CML agents according to methods of the present invention.
  • an animal manifesting CML is subjected to whole body hyperthermia, i.e., the body temperature of the animal is increased above normal, and a compound to be tested for anti-CML activity is administered prior to, during or after WBH.
  • the compound to be tested is prepared in a suitable pharmacological carrier, such as normal saline, in an amount appropriate for the particular test mammal.
  • the compound is injected intravenously into the mammal.
  • the effectiveness of the compound as an anti-CML agent is determined by analyzing for cell death of CML cells, particularly BCR/abl transformed cells; analysis for tyrosine kinase activity; analysis for PI 3- kinase activity (see e.g., Skorski, T., et al., Blood, 1995, 86:2, 726-36); analyzing for Philadelphia positive cells; using fluorescence in situ hybridization (FISH) to find positive cells, or by other methods known in the art.
  • FISH fluorescence in situ hybridization
  • the present invention is further illustrated by the following Examples.
  • the Examples are provided to aid in the understanding of the invention and are not construed as a limitation thereof.
  • Hematopoietic cell lines BaF-3 (BaF), 32D and FDCP were maintained in RPMI containing 10% FCS and 10-20% conditioned medium from IL-3 producing cell line, WEHI-3B. (Palacios R, et al., Cell 1985; 41 : 727-734).
  • the p210-BCR/abl cDNA was expressed in parental BaF and 32D cells by electroporation and selected for G418 resistance to generate transformed stable cell lines BaFp210 and 32D-p210, respectively, as described.
  • Parental and BCR/abl transformed FDCP cell lines were obtained from Jaqueline Pierce (NCI, Bethesda, MD).
  • Parental and NIH3T3 fibroblast cell lines which express the following constructs: gr ⁇ grp210 BCR/abl, SH3 deleted c-abl ( ⁇ XB), and v-abl were grown in DMEM supplemented with 10% calf sera as described. (Daley GQ, et al., Science 1987; 237: 532-535; Varticovski L, et al., Mol Cell Biol 1991 ; 1 1: 1 107- 1 1 13). Cells were deprived of serum and conditioned media for 14- 16 hours prior to harvest and whole cell lysates (WCL) were prepared as described.
  • WCL whole cell lysates
  • the cell lines used herein contain secondary mutations which enable the cells to survive without BCR/abl activity.
  • the effects of the mutations on these cell lines, e.g., immortality, are known. See e.g., Klucher, K.M. et al., Blood, 1998, 9:3927-3934.
  • Anti-phospho-tyrosine 4G10 Upstate Biotechnology, Inc. Lake Placid, NY; Monoclonal and polyclonal anti-abl antibody, AB3 and AB l, respectively (Calbiochem-Novatech, Cambridge, MA).
  • Anti-SHPl, -SHP2 and -PTP1B antibodies were kindly provided by Benjamin Neel (Harvard Institutes of Medicine, HMS, Boston, MA).
  • Goat anti-mouse horseradish peroxidase (HRP) conjugated and goat anti-rabbit HRP conjugated second antibodies were from New England Biolab (Beverly, MA) and Promega (Madison, WI), respectively.
  • D-PBS Dulbecco phosphate-buffered saline
  • lysis buffer containing 1% NP-40 50 mM HEPES pH 7.5, 150 mM sodium chloride, 50 mM sodium fluoride, 5 mM sodium orthovanadate, 0.5 mM EGTA, 10% glycerol and protease inhibitors (leupeptin and aprotinin at 10 ⁇ g/ml, pepstatin at 5 ⁇ g/ml and PMSF at 0.5 mM).
  • WCL were incubated with the appropriate antibodies for 2 hours at 4°C followed by addition of protein A/Sepharose for 45 minutes. Immune complexes were isolated by centrifugation, and washed with PBS, 2x with TNE (10 mM Tris/HCl, pH 7.4, 100 mM NaCI, 1 mM EDTA) and PBS. Immune precipitates were immediately used for enzyme assays or denatured by boiling in SDS gel loading buffer.
  • proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane.
  • Membranes were blocked in PBS- T (0.2% Tween-20, 7% nonfat dry milk, and 2% BSA) for 1 hour, probed with specific antibodies and incubated with HRP-conjugated second antibodies. Immune reactive bands were detected using chemiluminescence (ECL, Amersham, Arlington Heights, IL).
  • chemiluminescence ECL, Amersham, Arlington Heights, IL
  • stripping membranes were incubated for 10 min either in stripping buffer (150 mM NaCI, 10 mM Tris- HC1, pH 2.3) or in Ponceau-S (Sigma) for 20 minutes at room temperature, washed extensively and reprobed with a different antibody.
  • PI 3-kinase activity in anti-phospho-tyrosine immunoprecipitates was assayed using phosphatidylinositol phosphate (PI) and PI 4,5-P 2 as substrates.
  • PI phosphatidylinositol phosphate
  • PI 4,5-P 2 PI 4,5-P 2
  • the cells were incubated at different temperatures as indicated, harvested, washed once with D-PBS at appropriate temperature and lysed in phosphatase assay buffer (50 mM HEPES; pH 7.5, 150 mM NaCI, 1% (v/v) Triton X-100, 1 mM EDTA, 10% glycerol, 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin, 1 mM benzamidine, 0.5 mM PMSF).
  • phosphatase assay buffer 50 mM HEPES; pH 7.5, 150 mM NaCI, 1% (v/v) Triton X-100, 1 mM EDTA, 10% glycerol, 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin, 1 mM benzamidine, 0.5 mM PMSF).
  • Protein-tyrosine kinase substrate raytide (Oncogene, Cambridge, MA) was phosphorylated in the presence of Mg-[ ⁇ 32 P]-ATP using purified recombinant Abl kinase (Oncogene, Cambridge, MA) as per manufacturer's instructions. The products were precipitated by 10% ice-cold TCA containing 0.5% BSA, dried and redissolved in the assay buffer. Protein tyrosine phosphatase activity in the presence or absence of 0.5 mM Na-orthovanadate was measured as described. (LaMontagne KRJ, et al., Mol. Cell Biol. 1998; 18: 2965-2975).
  • reaction mixture containing 100,000 cpm of labeled raytide and WCL or immunoprecipitated FTP- IB used as a positive control was incubated at 30°C for 20 min. Reactions were terminated by addition of 375 ⁇ l of cold 4% activated Norit and centrifuged at 4°C for 8 min at 14,000 rpm. 200 ⁇ l of clear supernatant was removed and counted using beta scintillation counter to determine the amount of 32 P released from substrate. Phosphatase activity was calculated as cpm of 32 P released/hr/mg protein.
  • the p210 BCR/abl gene product was immunoprecipitated from BaFp210 cells using 1 mg WCL protein/assay and 5 ⁇ l of anti-Abl monoclonal antibody at 4°C for 2-3 hours. Immune complexes were isolated on Protein A/Sepharose beads (Sigma, St Louis, MO), washed three times with lysis buffer and once with ice-cold kinase buffer (40 mM HEPES; pH 7.4, 20 mM MnCl 2 , 20 mM MgCl 2 , 1 mM DTT and 0.2% Triton X-100).
  • Immunoprecipitated BCR/abl on beads was mixed with 25 ⁇ l of kinase buffer and preincubated for 30 min at different temperatures as indicated. The reaction was started by addition of 2 ⁇ g or raytide and 10 ⁇ Ci of [ ⁇ 32 P]- ATP and 25 ⁇ M cold ATP for 30 min. Reactions were terminated by transference of the tubes to ice-cold water bath and centrifugation at 12,000g for 5 min. 20 ⁇ l of supernatant was spotted on a phosphocellulose paper (p81 , Whatman) and remainder of the reaction mixture was boiled in SDS gel loading buffer, resolved by SDS-PAGE gel and exposed for autoradiography.
  • the autophosphorylation of BCR/abl was measured by excising the band from the gel and counting using a beta scintillation counter.
  • the purified recombinant Abl kinase activity was measured by addition of 2 ⁇ g raytide as substrate and processed as above. The filters were washed three times with 0.5% phosphoric acid, air dried and counted using a beta scintillation counter.
  • EXAMPLE 1 Temperature shift to 39° °C reduces tyrosine-phosphorylated proteins in BCR/abl transformed hematopoietic cells.
  • BCR/abl tyrosine kinase activity plays a critical role in BCR/abl mediated leukemogenesis and blocks apoptosis of hematopeietic cells used by cytokine removal and DNA damaging agents.
  • tyrosine kinase activity of p210 BCR/abl expressed in IL-3 dependent hematopoietic cells is downregulated at 39°C and have investigated the ability of BCR/abl to block apoptosis under these conditions.
  • BCR/abl transformed hematopoietic cells were grown in 10% FCS at 37 to 39°C for 16-72h.
  • Tyrosine phosphorylated proteins are observed in cells expressing BCR/abl cultured at 37°C ( Figure 1 , odd lanes) but not at 39°C (even lanes).
  • BCR/abl tyrosine kinase activity is dramatically reduced in hematopoietic cells maintained in culture at 39°C.
  • the temporal inactivation of BCR/abl tyrosine kinase provides an important model for characterization of mechanisms signal transduction by BCR/abl and permits novel approaches for designing treatment modalities for CML.
  • Figure 1 shows the downregulation of BCR/abl tyrosine kinase at 39°C in hematopoietic cell lines.
  • Figure 1A shows parental BaF (lanes 1, 2), hematopoietic cells expressing wt p210 BCR/abl, BaFp210 (lanes 3, 4), 32Dp210 (lanes 5,6) and FDCPp210 (lanes 7,8). The cells were grown at 37°C (odd lanes) and shifted to 39°C (even lanes) for 16 - 18 h. 50 ⁇ g of WCL protein was fractionated on SDS-PAGE gel, transferred onto nitrocellulose membranes and probed with anti-phospho-tyrosine antibodies. Position of the molecular weight markers is indicated on the right.
  • BCR/abl and transformation ability of Abl kinase are influenced by specific structural motifs which determine protein- protein interactions.
  • the reduction in BCR/abl induced tyrosine phosphorylation of intracellular substrates could be due to a decrease in the BCR/abl intrinsic kinase activity and changes in the interaction with potential substrates.
  • Figure 2 shows the downregulation of BCR/abl tyrosine kinase at 39°C is not influenced by BCR/abl structural motifs and is evident only when expressed in hematopoietic cells.
  • Figure 2 A shows a graphic representation of wt and mutants of BCR/abl and Abl. The position of various structural motifs is indicated.
  • G418 resistant mass transfected cells were used for all experiments and cell lines were examined for growth factor independence and expression of BCR/abl and Abl.
  • FIG. 2B shows that BCR/abl structural domains do not affect the kinase activity at 39°C.
  • BaF cells which express different BCR/abl or c-abl mutant proteins were grown as described under legends to Figure 1 at 37°C (odd lanes) or at 39°C (even lanes) for 16 - 18 h.
  • 50 ⁇ g of WCL protein was fractionated on SDS-PAGE gel, transferred onto nitrocellulose membranes and probed with anti-phospho-tyrosine antibodies. Position of the molecular weight markers is indicated on the right.
  • NIH3T3 fibroblasts expressing g ⁇ g-BCR/abl or v-abl were grown as indicated at 37°C or at 39°C for 24 h.
  • Parental NIH3T3 fibroblasts were induced to quiescence for 48, grown for additional 24 h at 37°C or 39°C and stimulated with 20ng/ml of PDGF-BB for 10 min.
  • 50 ⁇ g of WCL protein was used for western blot with anti- phospho-tyrosine antibodies. Position of molecular weight markers is indicated on the right.
  • EXAMPLE 3 Effect of temperature on Abl and BCR/abl tyrosine kinase activity in vitro.
  • kinase activity of immunoprecipitated p210 BCR/abl was tested at different temperatures using tyrosine kinase substrate, raytide.
  • Figure 3 shows the effect of temperature on p210 BCR/abl and recombinant purified Abl kinase.
  • p210 BCR/abl was immunoprecipitated from BaFp210 cell lysate and used for in vitro kinase activity.
  • BCR/abl on Protein A/Sepharose beads was preincubated for 30 min at different temperatures ranging from 4 - 39°C.
  • the kinase reactions were carried out at respective temperatures for additional 30 min, and kinase activity determined by the rate of 32 P incorporation into raytide substrate.
  • Data represents one of three independent experiments.
  • p210 BCR/abl in vitro kinase activity was maximal between 24°-28°C and reduced by 80% and 93% at 37°C and 39°C, respectively.
  • Figure 3B shows the effect of temperature on autophosphorylation of immunoprecipitated BCR/abl protein. After removing the clear supernatant as described in panel A, remaining samples were boiled in 1XSDS gel loading buffer, proteins fractionated on SDS-PAGE gels and dried gels were exposed for autoradiography. Data represents one of three independent experiments. Maximal autophosphorylation of BCR/abl occurred at 28°C (Fig 3B, lane 4) which was markedly reduced at 39°C to the levels observed at 4°C (Fig 3B, lane 7).
  • Figure 3 shows the effect of temperature on the kinase activity of purified recombinant Abl.
  • the temperature effect on purified Abl kinase was determined using GST-Crk as substrate. Kinase reactions were carried out at indicated temperatures as described under panel A. Each reaction contained 2 ⁇ g of GST-Crk and 2 units of Abl kinase. Data represents one of three independent experiments. Abl kinase activity was similar to that of p210 BCR/abl and was almost undetectable at 39°C (Fig 3C).
  • Figure 4 shows that expression or activity of protein tyrosine phosphatases in hematopoietic cell specific is not altered by temperature shift.
  • Figure 4 A shows the expression of FTP- IB, SH-PTPl and SH-PTP2 protein tyrosine phosphatases in BaFp210 cells at 39°C.
  • BaF (lane 1) or BaFp210 cells were grown at 37°C (lane 2) and shifted to 39°C (lanes 3 - 8) for 24 h.
  • cells were harvested and 50 ⁇ g of WCL protein was fractionated on SDS-PAGE gel, transferred onto nitrocellulose membranes and probed with anti-PTPlB, SH-PTPl or SH-PTP2 antibodies.
  • Expression of FTP1B, SH-PTPl and SH-FTP2 remained constant at 39°C ( Figure 4A). The apparent decrease in protein levels of SH-PTPl was not reproduced in other experiments.
  • Figure 4 B shows the total cellular protein tyrosine phosphatase activity is not altered in BaFp210 cells grown at 39°C.
  • Total FTP activity from equal quantities of lysate proteins ( 1 - 2 ⁇ g) from BaF or BaFp210 cells grown at indicated temperatures was determined using Abl kinase phosphorylated raytide as a phosphate donor.
  • Total phosphatase activity in whole cell extracts from cells grown at different temperatures overnight also showed no measurable difference between 37°C and 39°C (Fig 4B).
  • EXAMPLE 5 The effect of downregulation of BCR/ abl tyrosine kinase activity on PI 3 kinase activity and cell growth.
  • PI 3-kinase Activation of PI 3-kinase has been shown to correlate with tyrosine kinase activity and survival of BCR/abl transformed hematopoietic cell lines and CML cells (Jain SK, et al., Blood 1996; 88: 1542-1550; Jain SK, et al., Oncogene 1997; 14: 2217-2228; Skorski T, et al., Blood 1995; 86: 726-736) whereas expression of BCR/abl in NIH3T3 fibroblasts does not lead to accumulation of PI 3-kinase lipid products. (Varticovski L, et al., Nature 1989; 342: 699-702). The effect of temperature on PI 3-kinase activity was analyzed in cells shifted to 39°C and during their recovery at 37°C.
  • Figure 5 shows that the down-regulation of PI 3-kinase activity by temperature shift at 39°C does not correlate with proliferation of hematopoietic cells.
  • Figure 5 A shows that temperature-induces a reversible downregulation of PI 3-kinase.
  • BaFp210 cells grown at 39°C for 18 h (lane 1) were returned to 37°C for indicated time (lanes 2 - 5) or grown at 37°C for 18 h (lane 6).
  • PI 3-kinase activity was measured in anti-phospho-tyrosine immunoprecipitates from cells grown at indicated temperatures using PI and PIP 2 as substrates. Data represents one out of four independent experiments.
  • FIG. 5 B shows that downregulation of BCR/abl activity by temperature does not affect cell growth or IL-3 independence.
  • BaF and BaFp210 cells were cultured at 5 x 10 4 cells/ml at indicated temperatures (open circles: 37°C, closed circles: 39°C).
  • Parental BaF cells were grown in the presence of 20% conditioned media from WEHI-3B cells. Number of live and dead cells were counted every 24 h by trypan blue exclusion. Data represents one out of six independent experiments.
  • EXAMPLE 6- Treatment of bone marrow or stem cells with ex vivo hyperthermic exposure.
  • the bone marrow of a patient having CML is removed by known methods in the art.
  • the marrow is removed at the time of remission prior to treatment with chemotherapy or radiation therapy.
  • the bone marrow is subjected to a temperature of 39°C, for about 16 to 24 hours, to kill any residual tumor cells.
  • an anti-CML agent such as Interferon or abl kinase inhibitors, is added to the bone marrow.
  • the anti-CML agent may be added prior to, during or after, hyperthermic treatment.
  • the bone marrow is checked before and after using methods known in the art, such as RT-PCR, FISH, for the absence of tumor cells or for the presence of the Philadelphia chromosome.
  • the tumor free bone marrow is then transplanted back into the patient, according to known methods.
  • EXAMPLE 7 Methods of screening compounds for use as anti-CML agents in conjunction with hyperthermia.
  • An animal e.g., mouse or rat, manifesting CML is anesthetized according to known methods.
  • the mouse is subjected to increased external temperatures by any one of a number of methods such as high temperature hydrotherapy with immersion, hot warming blankets and insulation, forms of radiant energy and exracorporeal circulation. For example, the mouse is subjected to radiant heat.
  • the air surrounding the mouse is humidified.
  • the animal is monitored to evaluate body temperatures and heart rate.
  • the mouse is subjected to elevated temperatures until the desired temperature is reached.
  • the animal is removed and covered with blankets. While covered, the higher body temperature is maintained for the period of time specified in each treatment plan.
  • the heat-retaining blankets are removed and body cooling begins.
  • the compound to be tested for anti-CML activity is administered prior to, during or after WBH.
  • the compound to be tested is prepared in a suitable pharmacological carrier, such as normal saline, in an amount appropriate for the particular test mammal.
  • a suitable pharmacological carrier such as normal saline
  • the compound is injected intravenously or intraperitoneally into the mammal.
  • the effectiveness of the compound as an anti-CML agent is determined by methods known in the art.

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Abstract

The present invention relates to a method of reducing tumor cells in bone marrow prior to transplantation into a patient suffering from myelogenous leukemia, particularly chronic myelogenous leukemia (CML), comprising subjecting the bone marrow to a temperature above 37 °C. The present invention also relates to a method for treating myelogenous leukemia comprising subjecting a patient having the disease to whole-body hyperthermia (WBH), wherein the body temperature of the patient, or the animal is raised to a temperature in the range of at least about 39 °C. The invention further relates to a method of screening compounds for treating myelogenous leukemia.

Description

TREATMENT OF MYELOGENOUS LEUKEMIA
BACKGROUND OF THE INVENTION
1. Field of the Invention The present invention relates to method for treating myelogenous leukemia.
2. Background
Leukemia is a malignant disease of the bone marrow and blood. The common types of leukemia are divided into myelogenous leukemia and lymphocytic leukemia. Lymphocytic leukemia involves uncontrolled growth of lymphocytes in the marrow, leading invariably to an increase in the concentration of lymphocytes in the blood. Lymphocytes are cells that mediate the specificity of the immune response.
There are about 10,000- 15,000 new cases of myeloid (also called non- lymphocytic or granulocytic) leukemia in the U.S. per year (Cancer Facts & Figures, American Cancer Society, 1987). There are two major forms of myeloid leukemia: acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML, also known as chronic myeloid leukemia). CML is the most common type of leukemia.
Despite treatment with chemotherapy, long-term survival in patients with AML is less than 10-20% (Clarkson et al., CRC Critical Review in Oncology/ Hematology 4, 221 (1986)), and survival with CML and related diseases such as chronic myelomonocytic leukemia (CMML) , chronic monocytic leukemia (CMMOL) and myelodysplastic syndrome (MDS) is even lower.
CML is characterized cytologically by the Philadelphia (Ph) chromosome, which results from the translocation of the Abelson (abl) oncogene on chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22. The DNA from chromosome 9 contains most of the proto- oncogene designated c-abl. The break in chromosome 22 occurs in the middle of the bcr gene. The resulting Ph chromosome has the 5' section of bcr fused with most of c-abl. The result of this translocation is the expression of a constitutively active cytoplasmic protein-tyrosine kinase activity, BCR/abl. (Clark, S.S., et al., (1989) Annu. Rev. Med., 40; 113- 122; Groffen, J., et al., (1984) Cell, 36; 93-99).
The BCR/abl oncogene most frequently encountered in human leukemias encodes frequently two proteins, p l85 (frequently referred to in the literature as p l90), with smaller BCR domain (amino acids 1-426) and p210, with a larger BCR domain (amino acids 1-902 or 926). p 185 is associated with acute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML) and p210 is found in almost all cases of chronic myelogenous leukemia (CML). (Ramakrishnan L, et al., Biochim Biophys Ada 1989; 989 (2): 209-224; Wiedemann LM, et al., Blood 1988; 71: 349-355; Daley GQ, et al., Science 1990; 247: 824-830). Expression of the 210 kDa form of BCR/abl leads to transformation of hematopoietic cells and is a hallmark of CML. Activated tyrosine kinase of abl is required for transformation of hematopoietic cells and triggers multiple signaling pathways. The transforming capacity of BCR/abl is based in its enhanced tyrosine kinase activity. (Daley GQ, et al., Proc Natl Acad Sci, USA 1988; 85: 9312-9316; Lugo TG, et al., Sάence 1990; 247: 1079- 1082; Cortez D, et al., Mol Cell Biol 1995; 15: 5531-5541). Several intracellular signaling intermediates, including p21ras and PI 3-Kinase, are activated in BCR/abl transformed cells. (Mandanas, R.A., et al., (1993) Blood, 82; 1838-1847; Pendergast, A. M., et al., (1993) Cell, 75; 175- 185; Varticovski, L., et al., (1991) Mol. Cell. Biol., 1 1 ; 1107- 1113.)
BCR/abl expression in cytokine-dependent murine hematopoietic cell lines leads to growth factor independence (Daley GQ, et al, Proc Natl Acad Sci, USA 1988; 85: 9312-9316; Hariharan IK, et al., Oncogene 1988; 3: 387- 399) and protection of cells from apoptosis induced by irradiation or cytotoxic agents. (Cortez D, et al., Mol Cell Biol 1995; 15: 5531-5541; Nishϋ K, et al., Oncogene 1996; 13: 2225-2234; Cortez D, et al., Oncogene 1996; 13 ( 12): 2589-2594; Cortez D, et al., Oncogene 1997; 15: 2333-2342). Whether CML progenitor cells are growth factor independent in addition to resistant to apoptotic stimuli is currently under investigation. (Albrecht T, et al., BrJ Haematol 1996; 95: 501-507; Bedi A, et al., Blood 1995; 86: 1148-1158; Cambier N, et al., Oncogene 1998; 16: 349-357; Maguer-Satta V, et al., Oncogene 1998; 16: 237-248).
Temperature-sensitive constructs of υ-abl (Engelman A, et al., Proc Natl Acad Sci, USA 1987; 84: 8021-8025; Engelman A, et al., J Virol 1990; 64: 4242-4251) and BCR/abl have been used (LaMontagne KRJ, et al., MolCellBiol 1998; 18: 2965-2975; Carlesso N, et al., Oncogene 1994; 9: 149- 156; Kabarowski JHS, et al., EMBO J 1994; 13: 5887-5895) in order to test the impact of BCR/abl expression. This approach may alter substrate recognition and specificity and demonstrate that aberrant temperature could affect signaling pathways other than those which depend on BCR/abl. (Cortez D, et al., Oncogene 1997; 15: 2333-2342).
Fever is a key feature of infection and inflammation, and is a common feature in autoimmune diseases. In vitro hyperthermia has been shown to potentiate immune responses to IL- 1 , IL-2 and immune response to antigen including enhanced antigen processing. (Cristau B, et al., J Immunol 1994; 152: 1546- 1556; Duff GW, et al., Clin Res 1982; 30: 694-699; Kappel M, et al., Immunology 1991; 73: 304-308; Huang YH, et al., Clin Exp Immunol 1996; 103: 61-66). It has been proposed that because lymphocytic leukemia is a disease of the immune system, whole-body hyperthermia would be a useful treatment regimen. (Robins et al., J. Clin. Oncol. Vol. 2, No.9: 1050 (1984)). However, hyperthermia has not been considered as a treatment option for CML.
Resistance to chemotherapeutic treatment has been observed in CML cells. Despite the fact that the function of the bcr-abl fusion protein has been studied by different groups, drug resistance of the patient with CML to chemotherapeutic treatment remains an unsolved problem. Szczylik, et al., (Science, 253:562, 1991) indicated that antisense oligonucleotides to the bcr-abl breakpoint junction inhibited cell proliferation in patient-derived CML cells, and suggested using such antisense alone to treat CML patients. One treatment for CML in patients that do not respond to chemotherapy is a bone marrow transplant. A transplant is necessary because any treatment that removes all the BCR/abl tumor cells from the marrow also kills the stem cells which are known to express BCR/abl, leaving the patient with no ability to make new blood cells. An allogeneic bone marrow transplant is the transfer of stem cells from a donor to the recipient CML patient and autologenous transplant is the transfer of stem cells from CML patient in remission, after treatment, to the same patient. Thus, it would be desirable to have a method of killing tumor cells in bone marrow that minimizes the effect on stem cells. Skorski, T., et al., (J. Clin. Invest. 92: 194, 1993) also showed that treatment of Philadelphia leukemic cells, mixed 1: 1 with normal bone marrow cells, with a combination of a low dose of mafosfamide and antisense to the bcr-abl breakpoint junction, was effective in killing the leukemic cells while sparing a high number of the normal cells and at least part of the abl sequence. The authors also emphasized the importance of not including antisense to the bcr region in order to avoid perceived deleterious side effects. However, based on this therapeutic approach, therapeutic antisense oligonucleotides would have to be individually designed in order to be effective, since the breakpoint junction between bcr and abl appears to vary from patient to patient.
Therefore, there is a need to develop a generalized method for inducing apoptosis in a cell having a bcr-abl translocation which avoids the necessity of determining a patient's particular bcr-abl breakpoint nucleotide sequence in order to develop an effective therapeutic agent. There is also a need for treatments of bone marrow cells which kills the leukemic cells while minimizing the effect on the normal cells. The present invention provides such methods, and enhances the effect of therapeutic agents in treating CML.
SUMMARY OF THE INVENTION
The present invention provides an ex υivo method of reducing tumor cells in bone marrow or stem cells collected from a patient suffering from myelogenous leukemia, particularly chronic myelogenous leukemia (CML), prior to transplant into the patient. The method comprises subjecting the bone marrow or stem cells to a temperature above 37°C for a period of time sufficient to inactivate tumor cells contained therein. Preferably the temperature is raised within a range of about 38°C to about 42°C, preferably about 39°C, for a period time sufficient to deactivate tumor cells contained therein, preferably for about 2 to 72 hours, more preferably for about 12 to 24 hours and most preferably about 16 to 18 hours. These methods preferably further comprise administering at least one anti-tumor cell agent, e.g., anti-CML agent, to the bone marrow or collected stem cells prior to, during, or after the heat treatment. The term "stem cell preparations" as used herein includes preparations containing stem cells from bone marrow or peripheral blood.
The present invention further provides a method for treating myelogenous leukemia, particularly CML, comprising subjecting a patient having the disease to whole-body hyperthermia (WBH), wherein the body temperature of the patient is raised to a temperature above 37°C. Preferably the body temperature is raised within a range of about 39°C to about 42°C, preferably about 30°-40°C, for about 2 hours to about 24 hours, preferably about 16 to 18 hours. These methods are also useful for treating other diseases expressing the BCR/abl gene.
The present invention also provides a method for treating myelogenous leukemia comprising subjecting a patient having CML to whole-body hyperthermia (WBH), and administering at least one anti-CML agent to the patient prior to, during, or after the patient has undergone whole body hyperthermia. Anti-CML agents include conventional therapy of chemotherapy and biologic response modifiers. The treatment can be repeated at periodic intervals, e.g., about 48 to 72 hours, preferably about every 48 hours, for a period of 7 to 10 days, preferably for a week.
The present invention also provides a method of screening compounds for treating chronic myelogenous leukemia comprising subjecting an animal having CML to whole-body hyperthermia (WBH), wherein the body temperature of the animal is raised above its normal body temperature, administering at least one compound or composition to be evaluated for anti-CML properties to the animal prior to, during, or after the animal has undergone whole body hyperthermia and determining the effectiveness of the compound by evaluating the effect of the treatment on the animal's CML cells. This can be done by evaluating for materials produced by or associated with CML cells, such as BCR/abl tyrosine kinase activity, tyrosine phosphorylated proteins, PI 3-kinase activity, or by other methods including but not limited to cell number, cell survival and apoptosis.
The present invention also provides a method of downregulating abl kinase activity of BCR/abl and v-abl transformed hematopoietic cells comprising subjecting the cells to temperatures above 37°C. This method results in reduction of tyrosine phosphorylated substrates in hematopoietic cells.
These methods of treating stem cells, methods of treating patients and methods of screening are also useful for treating other diseases involving cells which express BCR/abl gene products.
Other aspects of the invention are disclosed infra.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the downregulation of BCR/abl tyrosine kinase at
39°C in hematopoietic cell lines. Figure 1A shows parental BaF (lanes 1, 2), hematopoietic cells expressing wild type (wt) p210 BCR/abl, BaFp210 (lanes 3, 4), 32Dp210 (lanes 5,6) and FDCPp210 (lanes 7,8). Figure IB shows the time course of BCR/abl tyrosine kinase downregulation at 39°C. Figure 1C shows that temperature-induced downregulation of BCR/abl tyrosine kinase in CMb cells is potentially reversible.
Figure 2 shows the downregulation of BCR/abl tyrosine kinase at 39°C is not influenced by BCR/abl structural motifs and is evident only when expressed in hematopoietic cells. Figure 2A is a graphic representation of wt and mutants of BCR/abl and Abl. Figure 2B shows that BCR/abl structural domains do not affect the kinase activity at 39°C. Figure 2C shows BCR/abl, Abl and PDGF-R kinase activities are not downregulated in NIH3T3 fibroblasts by increased temperature. Figure 3 shows the effect of temperature on p210 BCR/abl and recombinant purified Abl kinase. Figure 3 A shows a graph of kinase activity determined by the rate of 32P incorporation into raytide substrate. Figure 3B shows the effect of temperature on autophosphorylation of immunoprecipitated BCR/abl protein. Figure 3C shows the effect of temperature on the kinase activity of purified recombinant Abl.
Figure 4 shows that expression or activity of protein tyrosine phosphatases in hematopoietic cell species is not altered by temperature shift. Figure 4A shows expression of FTP- IB, SH-PTPl and SH-PTP2 protein tyrosine phosphatases in BaFp210 cells at 39°C. Figure 4B shows is a graph showing total cellular protein tyrosine phosphatase activity is not altered in BaFp210 cells grown at 39°C.
Figure 5 shows that down-regulation of PI 3-kinase activity by temperature shift at 39°C does not correlate with proliferation of hematopoietic cells. Figure 5A shows temperature-induction of a reversible downregulation of PI 3-kinase. Figure 5B shows that downregulation of BCR/abl activity by temperature does not affect cell growth of the BaFp210 cell line or IL-3 independence.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have surprisingly discovered that BCR/abl tyrosine kinase activity is dramatically reduced in hematopoietic cells maintained in culture at temperatures above normal body temperature.
Exposure of BCR/abl and v-abl transformed hematopoietic cells or purified proteins to 39°C downregulates Abl kinase activity and results in a marked reduction in tyrosine phosphorylated substrates in hematopoietic cells. The expression of BCR/abl is unaffected and tyrosine kinase activity can be recovered when cells are shifted back to 37°C.
The down regulation of kinase activity increases the number of tumor cells killed during this period, providing an ex vivo method to reduce tumor cells in bone marrow to be used in treating patients having CML or in stem cell preparations from peripheral blood. In one method of the present invention used in autologous transplants, the patient's own bone marrow or collected stem cells are subjected ex-vivo to a temperature above 37°C for a period of time sufficient to kill any residual tumor cells. To increase the number of cells killed, an anti-tumor cell agent, e.g., anti-CML agent, is preferably added to the bone marrow or stem cell preparation. The anti-CML agent may be added prior to, during or after hyperthermic treatment. The bone marrow or stem cell preparation is subjected to the increased temperature for a period of time that is sufficient to kill the tumor cells, while minimizing the adverse effects on normal cells in the marrow or preparation. A preferred range of time for treatment is from about 2 to 72 hours, more preferably about 10 to 24 hours, most preferably about 16 hours.
Similarly, blood, e.g., collected peripheral blood and blood products may be treated with the same ex vivo treatment to kill tumor cells prior to transfusion. Such treatments can be used on the patient's own blood or blood products to eliminate tumor cells or a donor's blood or blood products. As aforesaid, in such a method, the blood or blood product is subjected to a temperature above 37°C to kill tumor cells. To increase the number of cells killed, an anti-CML agent may be added to the blood or blood product, prior to, during or after hyperthermic treatment.
Anti-CML agents useful in the practice of the present invention include conventional chemotherapy and biologic response modifiers, e.g., interferon. See, Katarjian, H.P., et al., Blood, 82: 691-703, 1993. Additional anti-CML agents include tyrosine kinase inhibitors that can be used to block BCR/abl tyrosine kinase activity. Such inhibitors include, for example, tyrphostins, 2-phenylaminopyrimidine derivatives, pyrazolpyrimidines, banzopyranones and benzothiopyranones, herbimycin A, and glendamycin. See, Buchdunger, E., et al., (1996) Cancer Res., 56; 100- 104; Anafi, M., et al., (1993) Blood, 82; 3524-3529; Geissler, J.F., et al., (1992) Cancer Res., 52; 4492-4498; Mohammadi, M., G., M., et al., (1997) Science, 276; 955- 960; Druker, B.J., et al., (1996) Nature Med., 2; 561-566; Fotsis, T., et al., (1993) Proc. Natl. Acad. Sci., USA, 90; 2690-2694; Hanke, J.H., et al., (1996) J. Biol. Chem., 271; 695-701; James, G.L., et al., (1993) Science, 260; 1937- 1942; Kaur, G., et al., ( 1994) Anti-Cancer Drugs, 5; 213-222; Peterson, G. et al.,. (1991) Biochem. Biophys. Res. Comm., 179; 661-667. In another method of treating a patient having CML, the patient is subjected to whole-body hyperthermia (WBH). In this method, the body temperature of the patient is raised to a temperature in the range of about 38°C to about 42°C, preferably about 39°C, for about 2 hours to about 24 hours, preferably about 16 hours. The treatment can be repeated at periodic intervals, e.g., 16 hours, every 2-3 days for a week, to eliminate the tumor cells. Anti-CML agents can be administered to the patient prior to, during, or after the patient has undergone whole body hyperthermia.
Whole body hyperthermia is a treatment in which the body temperature is raised from 37°C up to 42°C. Hyperthermia has the ability to selectively kill some heat sensitive malignancies. In this case, hyperthermia can be combined with other treatments (for example, chemotherapy) to increase their effect. Although hyperthermia was first introduced as a treatment for cancer more than one hundred years ago, its use continues to be actively investigated.
Body temperature can be raised by a number of mechanisms which are known in the art. For example, techniques include paraffin wax baths, high temperature hydro therapy with immersion, hot warming blankets and insulation, forms of radiant energy and extracorporeal circulation. One whole body hyperthermia device, called the "Aquatherm", utilizes a radiant heat technology. The radiant heat produced by the Aquatherm produces heat while air temperatures are elevated only minimally. To prevent heat losses from the evaporation of perspiration, which would slow heating, the air in the device is humidified additionally. This device uses a water tight cylindrical coil through which heated water can be pumped while maintaining low pressure. Design features for this device include a counter- current flow pattern to maintain temperature homogeneity. A humidification system maintains humidity within the chamber. The Aquatherm controls radiant heat exchanges to supplement metabolic heat production while minimizing evaporative heat losses. The subject to be treated lies on a stretcher which is mounted on rails to allow easy movement in and out of the device. A non-conductive plastic netting is provided inside the heating chamber to prevent patient contact with the inner heat-radiating surface of the chamber. An industry standard 40 W light is incorporated into the rear wall of the device to maximize patient observations during treatments. Two thermal insulating plexiglass doors are provided at the head of the apparatus. These doors allow the patient's head to remain outside the heating chamber at all times; they also allow for patient observation. A soft collar is incorporated into the door design which fits around the neck of the patient. This apparatus is disclosed in U.S. Pat. No. 5,713,941, the disclosure of which is herein incorporated by reference.
At the start of the actual treatment, various monitoring devices are attached to the patient. These monitors assist the hyperthermia team in ensuring patient comfort and safety. Monitoring includes body temperatures, heart rate, blood pressure, urine production, breathing rate, and state of consciousness. An IN. is started for the purpose of administering fluids and medications. After the necessary pretreatment preparations are made, the patient's body, except for the head and neck, remains in the Aquatherm device for approximately 1.5 hours in order to achieve the desired temperature. At this time the patient is removed from the device and covered with blankets. While covered, the higher body temperature is maintained for the period of time specified in each treatment plan. During the heating and treatment phases, medications are administered for patient comfort. The patient will feel drowsy during the treatment but is able to talk with the hyperthermia team. At the conclusion of the treatment, the heat- retaining blankets are removed and body cooling begins. Monitoring of the patient continues until body temperature returns to the normal range. Generally, treatment duration from start to finish is less than 16 hours.
The present inventors demonstrate that tyrosine kinase activity of wild type BCR/abl is temperature sensitive in vitro, when expressed in hematopoietic cells. Tyrosine phosphorylation of cellular substrates and PI 3-kinase activity in BCR/abl and v-abl transformed hematopoietic cells are abrogated by overnight exposure to 39°C without change in protein expression. This effect is reversible, and phosphorylation of intracellular proteins is fully restored at 37°C within 4-12 hours. Temperature-induced downregulation of BCR/abl tyrosine kinase activity in vivo is apparent after 16 h, whereas 5 min exposure of purified recombinant Abl kinase to 39°C is sufficient to decrease its activity in vitro. Thus, Abl kinase activity is intrinsically sensitive to a 2°C shift above normal body temperature. While the inventors do not intend to be bound by theory, it is postulated that the action of protein-tyrosine phosphatases and the interaction with intracellular proteins determines protein folding which is responsible for maintaining Abl tyrosine kinase activity and the level of tyrosine phosphorylated substrates for up to 16 hours.
The methods of the present invention are selective for abl kinase. Temperature-induced inhibition of Abl kinase activity in vivo in cell lines was detected only in hematopoietic cells and did not occur in NIH3T3 fibroblasts which express the same gene products. Activation of PDGF-R kinase was also not altered after prolonged incubation of NIH3T3 fibroblasts at 39°C. Downregulation of Abl kinase activity was not induced when expressed in NIH3T3 fibroblasts. Although the inventors do not intend to be bound by theory, this data supports the hypothesis that interaction with cell- specific factors facilitates a continued activation of Abl kinase in hematopoietic cells in spite of intrinsic sensitivity of the kinase to higher temperature.
It has been difficult to generate animal models which accurately reflect the biological phenotype of CML. Genetic instability has long been recognized as a main cause of progression of CML into acute leukemia during terminal stages of the disease associated with blast crisis. The present inventors have discovered that, in the absence of mutations, human wt BCR/abl expressed in hematopoietic cells is also sensitive to a 2°C increase in temperature which is in the range of hyperthermia associated with fever. In spite of significant decrease in tyrosine phosphorylated substrates in BCR/abl transformed hematopoietic cells maintained at 39°C and lack of PI 3-kinase activity, there was no decrease in growth factor independence. These results suggest that BCR/abl-induced genetic instability may account for secondary mutations which provide alternative mechanisms for cell survival and no longer require active BCR/abl. Alternatively, low levels of BCR/abl tyrosine kinase activity may be sufficient to maintain proliferation of these cells whereas higher levels of kinase activity are necessary for protection against apoptosis. A dose dependent hierarchy for BCR/abl induced biological effects in hematopoietic cells was previously proposed. ^ The increase in temperature may also have an independent effect on activation of other survival pathways which potentiate biological response to BCR/abl.
The present invention also relates to methods of screening compounds for use as anti-CML agents. Although CML does not spontaneously occur in animals, animal models for studying CML are now known in the art. (See e.g., Daley, G.Q., et al., Science, 1990, 247:4944, 824-30; Daley, G.Q., et al., Proc. Natl. Acad. Sci., 1991 , 88:24, 11335-8; Ilaria, R.L., et al., Blood, 1995, 86: 10, 3897-904). These animal models can be used to develop and test potential compounds for use as anti-CML agents according to methods of the present invention. In such a method, an animal manifesting CML is subjected to whole body hyperthermia, i.e., the body temperature of the animal is increased above normal, and a compound to be tested for anti-CML activity is administered prior to, during or after WBH. The compound to be tested is prepared in a suitable pharmacological carrier, such as normal saline, in an amount appropriate for the particular test mammal. Preferably, the compound is injected intravenously into the mammal. The effectiveness of the compound as an anti-CML agent is determined by analyzing for cell death of CML cells, particularly BCR/abl transformed cells; analysis for tyrosine kinase activity; analysis for PI 3- kinase activity (see e.g., Skorski, T., et al., Blood, 1995, 86:2, 726-36); analyzing for Philadelphia positive cells; using fluorescence in situ hybridization (FISH) to find positive cells, or by other methods known in the art. This method is useful for screening new compounds as anti-CML agents as well as determining the effect of WBH on the effectiveness of existing therapeutic compounds.
The present invention is further illustrated by the following Examples. The Examples are provided to aid in the understanding of the invention and are not construed as a limitation thereof.
Cells:
Hematopoietic cell lines BaF-3 (BaF), 32D and FDCP were maintained in RPMI containing 10% FCS and 10-20% conditioned medium from IL-3 producing cell line, WEHI-3B. (Palacios R, et al., Cell 1985; 41 : 727-734). The p210-BCR/abl cDNA was expressed in parental BaF and 32D cells by electroporation and selected for G418 resistance to generate transformed stable cell lines BaFp210 and 32D-p210, respectively, as described. (Daley GQ, et al., Proc Natl Acad Sci, USA 1988; 85: 9312-9316; McLaughlin J, et al., Proc Natl Acad Sci, USA 1987; 84: 6558-6562). BaFp210-R1053K, BaFp210-ΔSH2, Bpl90, B-v-abl and B-c-abl-ΔSH3 stable cell lines were generated by electroporation of plasmid DNA encoding various isoform of abl into BaF3 cells. All DNA constructs were kindly provided by R. Van Etten (HMS, Boston, MA) and their structural features are shown in Figure 2. Parental and BCR/abl transformed FDCP cell lines were obtained from Jaqueline Pierce (NCI, Bethesda, MD). Parental and NIH3T3 fibroblast cell lines which express the following constructs: grαgrp210 BCR/abl, SH3 deleted c-abl (ΔXB), and v-abl were grown in DMEM supplemented with 10% calf sera as described. (Daley GQ, et al., Science 1987; 237: 532-535; Varticovski L, et al., Mol Cell Biol 1991 ; 1 1: 1 107- 1 1 13). Cells were deprived of serum and conditioned media for 14- 16 hours prior to harvest and whole cell lysates (WCL) were prepared as described. (Varticovski L, et al., Nature 1989; 342: 699-702) . Unlike diseased cells in patients suffering from CML, the cell lines used herein contain secondary mutations which enable the cells to survive without BCR/abl activity. The effects of the mutations on these cell lines, e.g., immortality, are known. See e.g., Klucher, K.M. et al., Blood, 1998, 9:3927-3934.
Antibodies:
Anti-phospho-tyrosine 4G10 (Upstate Biotechnology, Inc. Lake Placid, NY); Monoclonal and polyclonal anti-abl antibody, AB3 and AB l, respectively (Calbiochem-Novatech, Cambridge, MA). Anti-SHPl, -SHP2 and -PTP1B antibodies were kindly provided by Benjamin Neel (Harvard Institutes of Medicine, HMS, Boston, MA). Goat anti-mouse horseradish peroxidase (HRP) conjugated and goat anti-rabbit HRP conjugated second antibodies were from New England Biolab (Beverly, MA) and Promega (Madison, WI), respectively.
Immunoprecipitations and Western blotting:
Cells were maintained at 37°C and shifted to 39°C for different time intervals as indicated in Figure legends. Cells were washed in Dulbecco phosphate-buffered saline (D-PBS) prewarmed to a specific temperature and lysed in lysis buffer containing 1% NP-40 in 50 mM HEPES pH 7.5, 150 mM sodium chloride, 50 mM sodium fluoride, 5 mM sodium orthovanadate, 0.5 mM EGTA, 10% glycerol and protease inhibitors (leupeptin and aprotinin at 10 μg/ml, pepstatin at 5 μg/ml and PMSF at 0.5 mM). For immunoprecipitations, WCL were incubated with the appropriate antibodies for 2 hours at 4°C followed by addition of protein A/Sepharose for 45 minutes. Immune complexes were isolated by centrifugation, and washed with PBS, 2x with TNE (10 mM Tris/HCl, pH 7.4, 100 mM NaCI, 1 mM EDTA) and PBS. Immune precipitates were immediately used for enzyme assays or denatured by boiling in SDS gel loading buffer.
For western blotting, proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were blocked in PBS- T (0.2% Tween-20, 7% nonfat dry milk, and 2% BSA) for 1 hour, probed with specific antibodies and incubated with HRP-conjugated second antibodies. Immune reactive bands were detected using chemiluminescence (ECL, Amersham, Arlington Heights, IL). For stripping, membranes were incubated for 10 min either in stripping buffer (150 mM NaCI, 10 mM Tris- HC1, pH 2.3) or in Ponceau-S (Sigma) for 20 minutes at room temperature, washed extensively and reprobed with a different antibody.
PI 3-kinase assay:
PI 3-kinase activity in anti-phospho-tyrosine immunoprecipitates was assayed using phosphatidylinositol phosphate (PI) and PI 4,5-P2 as substrates. (Susa M, et al.,. J Biol Chem 1992; 267: 22951-22956). Reaction products were extracted and subjected to thin layer chromatography. 32P incorporated into PI 3,4,5-P3 (PIP3) was quantified by scintillation counting and results expressed as counts/min following background subtraction.
Protein-Tyrosine Phosphatase Activities:
The cells were incubated at different temperatures as indicated, harvested, washed once with D-PBS at appropriate temperature and lysed in phosphatase assay buffer (50 mM HEPES; pH 7.5, 150 mM NaCI, 1% (v/v) Triton X-100, 1 mM EDTA, 10% glycerol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM benzamidine, 0.5 mM PMSF). Protein-tyrosine kinase substrate, raytide (Oncogene, Cambridge, MA), was phosphorylated in the presence of Mg-[γ32P]-ATP using purified recombinant Abl kinase (Oncogene, Cambridge, MA) as per manufacturer's instructions. The products were precipitated by 10% ice-cold TCA containing 0.5% BSA, dried and redissolved in the assay buffer. Protein tyrosine phosphatase activity in the presence or absence of 0.5 mM Na-orthovanadate was measured as described. (LaMontagne KRJ, et al., Mol. Cell Biol. 1998; 18: 2965-2975). In brief, reaction mixture containing 100,000 cpm of labeled raytide and WCL or immunoprecipitated FTP- IB used as a positive control was incubated at 30°C for 20 min. Reactions were terminated by addition of 375 μl of cold 4% activated Norit and centrifuged at 4°C for 8 min at 14,000 rpm. 200 μl of clear supernatant was removed and counted using beta scintillation counter to determine the amount of 32P released from substrate. Phosphatase activity was calculated as cpm of 32P released/hr/mg protein.
In vitro Abl tyrosine kinase activity:
The p210 BCR/abl gene product was immunoprecipitated from BaFp210 cells using 1 mg WCL protein/assay and 5 μl of anti-Abl monoclonal antibody at 4°C for 2-3 hours. Immune complexes were isolated on Protein A/Sepharose beads (Sigma, St Louis, MO), washed three times with lysis buffer and once with ice-cold kinase buffer (40 mM HEPES; pH 7.4, 20 mM MnCl2, 20 mM MgCl2, 1 mM DTT and 0.2% Triton X-100). Immunoprecipitated BCR/abl on beads was mixed with 25 μl of kinase buffer and preincubated for 30 min at different temperatures as indicated. The reaction was started by addition of 2 μg or raytide and 10 μCi of [γ32P]- ATP and 25 μM cold ATP for 30 min. Reactions were terminated by transference of the tubes to ice-cold water bath and centrifugation at 12,000g for 5 min. 20 μl of supernatant was spotted on a phosphocellulose paper (p81 , Whatman) and remainder of the reaction mixture was boiled in SDS gel loading buffer, resolved by SDS-PAGE gel and exposed for autoradiography. The autophosphorylation of BCR/abl was measured by excising the band from the gel and counting using a beta scintillation counter. The purified recombinant Abl kinase activity was measured by addition of 2μg raytide as substrate and processed as above. The filters were washed three times with 0.5% phosphoric acid, air dried and counted using a beta scintillation counter. EXAMPLE 1 - Temperature shift to 39° °C reduces tyrosine-phosphorylated proteins in BCR/abl transformed hematopoietic cells.
Mechanisms of p210 BCR/ abl-initiated signal transduction using a temperature-sensitive mutant of BCR/abl have been described elsewhere. (Jain SK, et al., Blood 1996; 88: 1542-1550). While performing these experiments, BaF cells were used which are transformed by wt p210 BCR/abl.
BCR/abl tyrosine kinase activity plays a critical role in BCR/abl mediated leukemogenesis and blocks apoptosis of hematopeietic cells used by cytokine removal and DNA damaging agents. We discovered that the tyrosine kinase activity of p210 BCR/abl expressed in IL-3 dependent hematopoietic cells is downregulated at 39°C and have investigated the ability of BCR/abl to block apoptosis under these conditions. BCR/abl transformed hematopoietic cells were grown in 10% FCS at 37 to 39°C for 16-72h. Shift of BCR/abl transformed cells to 39°C for 16h resulted in decreased tyrosine kinase activity and, after 48h, in a G1/G0 arrest without an increase in apoptotic cells as compared to cells growing at 37°C. After 72h, the number of apoptotic cells grown at 39°C increased 10 fold as compared with control cells maintained at 37°C. Increased DNA degradation was apparent in cells grown at 39°C for 48h before apoptosis was evident by FACS analysis. Addition of IL-3 did not reduce apoptosis of cells grown at 39° or 37°C. To determine whether BCR/abl activity was sufficient to induce quiescent cells to reenter the cell cycle, cells were shifted to 37°C after 16h at 39°C. Activation of BCR/abl tyrosine kinase, decrease in DNA degradation and reduction in the number of apoptotic cells but not an increase in S phase cells were evident at 48-72h. These results indicate that temperature-induced downregulation of BCR/abl activity is sufficient to trigger apoptosis.
Tyrosine phosphorylated proteins are observed in cells expressing BCR/abl cultured at 37°C (Figure 1 , odd lanes) but not at 39°C (even lanes). Thus, BCR/abl tyrosine kinase activity is dramatically reduced in hematopoietic cells maintained in culture at 39°C. The temporal inactivation of BCR/abl tyrosine kinase provides an important model for characterization of mechanisms signal transduction by BCR/abl and permits novel approaches for designing treatment modalities for CML.
Figure 1 shows the downregulation of BCR/abl tyrosine kinase at 39°C in hematopoietic cell lines. Figure 1A shows parental BaF (lanes 1, 2), hematopoietic cells expressing wt p210 BCR/abl, BaFp210 (lanes 3, 4), 32Dp210 (lanes 5,6) and FDCPp210 (lanes 7,8). The cells were grown at 37°C (odd lanes) and shifted to 39°C (even lanes) for 16 - 18 h. 50 μg of WCL protein was fractionated on SDS-PAGE gel, transferred onto nitrocellulose membranes and probed with anti-phospho-tyrosine antibodies. Position of the molecular weight markers is indicated on the right.
It was found that the major cellular substrates of BCR/abl (Cbl and She) were not tyrosine phosphorylated in these cells grown at 39°C (Figure 1 A, lanes 3 and 4). Subsequent analysis showed that p210 BCR/abl transformed 32D cells, 32D-p210 (lanes 5 and 6) and FDCP cells, FDCP- p210 (lanes 7 and 8) grown at 39°C also have markedly reduced tyrosine phosphorylated proteins. These data suggest that wt BCR/abl tyrosine kinase activity is sensitive to temperature shift in the absence of mutations.
The time course of wt BCR/abl inactivation at 39°C was analyzed. BaFp210 cells were grown at 37°C and shifted to 39°C for 0.5- 16 h and examined for tyrosine phosphorylation of cellular substrates. Figure IB shows the time course of BCR/abl tyrosine kinase downregulation at 39°C. BaFp210 cells were grown at 37°C (lane 1) and shifted to 39°C for indicated time intervals (lanes 2 - 6); cells were harvested and 50 μg WCL protein was fractionated on SDS-PAGE gel, transferred onto nitrocellulose membrane and probed with anti-phospho-tyrosine antibodies (Upper Panel).
To determine whether reduced tyrosine phosphorylation of proteins at
39°C could be due to decline in BCR/abl expression, anti-abl Western blotting of the same membranes was performed. BCR/abl protein expression remained unaltered (Fig IB, bottom panel). In the lower panel, the membrane was stripped and reprobed with anti-abl antibodies. Position of the molecular weight markers is indicated on the right. As shown in Figure IB, the pattern of tyrosine phosphorylated proteins in cells maintained at 39 °C for up to 12 h was similar to that of cells grown at 37°C (lanes 1 and 5). However, after ± 16 h at 39°C, BaFp210 cells contained only a fraction of tyrosine-phosphorylated proteins (lane 6) which persisted when cells were maintained at this temperature for longer periods of time. Similar results were seen in cells expressing wt pi 90 BCR/abl (not shown).
To examine whether the downregulation of protein tyrosine phosphorylation at 39CC is reversible, BaFp210 cells were grown at 39°C for 16 h and shifted back to 37°C for different time intervals (Figure 1C). Figure 1 C shows that temperature-induced downregulation of BCR/ abl tyrosine kinase in BaFp210 cells is reversible. BaFp210 cells grown at 39°C for 18 h (lane 1) and shifted back to 37°C for indicated time (lanes 2 - 6). Cells were harvested and 50 μg WCL protein was fractionated on SDS-PAGE gel, transferred onto nitrocellulose membrane and probed with anti-phospho- tyrosine antibodies. Position of the molecular weight markers is indicated on the right. Anti-phospho-tyrosine western blots showed that protein- tyrosine phosphorylation recovered gradually over 12 h while expression of BCR/abl was again unchanged (not shown).
Taken together, these results demonstrate that tyrosine phosphorylation of cellular proteins in BCR/ abl- transformed hematopoietic cells is sensitive to relatively small increases in temperature seen in febrile states, i.e. about 39°C, whereas the stability and expression of p210 BCR/abl is not affected.
EXAMPLE 2 - Structural requirements and spedficity of BCR/abl downregulation upon temperature shift.
Leukemogenic potential of BCR/abl and transformation ability of Abl kinase are influenced by specific structural motifs which determine protein- protein interactions. The reduction in BCR/abl induced tyrosine phosphorylation of intracellular substrates could be due to a decrease in the BCR/abl intrinsic kinase activity and changes in the interaction with potential substrates. To test whether mutation of specific structural domains of BCR/abl or Abl has an effect on temperature sensitivity of the wt p210 and pl90 BCR/abl, BaF cells which express wt p210 BCR/abl were compared with cells which express the following mutants (Figure 2A): p210R1053K BCR/abl, which abolishes the functional interaction with the Abl SH2 domain (Figure 2B, lanes 3 and 4); ΔSH2-p210 BCR/abl which has a deletion of the SH2 domain, (Figure 2B, lanes 5 and 6); v-abl (Figure 2B, lanes 7 and 8), ΔSH3-c-αb., a transforming variant of c-abl which lacks the SH3 domain (Figure 2B, lanes 9 and 10), P190 BCR/abl and p210Y177F BCR/abl which fails to bind the adapter protein, GRB2 (not shown).
Figure 2 shows the downregulation of BCR/abl tyrosine kinase at 39°C is not influenced by BCR/abl structural motifs and is evident only when expressed in hematopoietic cells. Figure 2 A shows a graphic representation of wt and mutants of BCR/abl and Abl. The position of various structural motifs is indicated.
To avoid clonal variations, G418 resistant mass transfected cells were used for all experiments and cell lines were examined for growth factor independence and expression of BCR/abl and Abl. To examine the temperature sensitivity, cells were grown at 39°C overnight, WCL normalized to expression of Abl protein and analyzed by western blotting with anti- phospho-tyrosine antibodies.
Figure 2B shows that BCR/abl structural domains do not affect the kinase activity at 39°C. BaF cells which express different BCR/abl or c-abl mutant proteins were grown as described under legends to Figure 1 at 37°C (odd lanes) or at 39°C (even lanes) for 16 - 18 h. 50 μg of WCL protein was fractionated on SDS-PAGE gel, transferred onto nitrocellulose membranes and probed with anti-phospho-tyrosine antibodies. Position of the molecular weight markers is indicated on the right. As shown in Figure 2B, a decrease in tyrosine phosphorylated proteins was observed in all cell lines exposed to 39°C which was similar to the results obtained using cells which express wt p210 BCR/abl gene product (Figure 2B, lanes 1 and 2). These results suggest that temperature sensitivity of BCR/abl tyrosine kinase is independent of the BCR, SH2 or SH3 domains and may reside in the tyrosine kinase domain or other domains in Abl not tested. Previous reports indicate that BCR/abl signal transduction is uniquely adapted for hematopoietic cells: it is highly transforming for hematopoietic cells whereas it requires plasma membrane targeting for transformation of NIH3T3 fibroblasts. (Varticovski L, et al., Mol Cell Biol 1991 ; 1 1 : 1 107- 1113). To examine whether temperature-mediated sensitivity of Abl kinase is evident in NIH3T3 fibroblasts, the pattern of tyrosine phosphorylation in cells which express transforming variants of BCR/abl in NIH3T3 fibroblasts: gfαgBCR/abl and v-abl were compared. Figure 2C shows that BCR/abl, Abl and PDGF-R kinase activities are not downregulated in NIH3T3 fibroblasts by increased temperature. NIH3T3 fibroblasts expressing gαg-BCR/abl or v-abl were grown as indicated at 37°C or at 39°C for 24 h. Parental NIH3T3 fibroblasts were induced to quiescence for 48, grown for additional 24 h at 37°C or 39°C and stimulated with 20ng/ml of PDGF-BB for 10 min. 50μg of WCL protein was used for western blot with anti- phospho-tyrosine antibodies. Position of molecular weight markers is indicated on the right.
We found no difference between cells which are grown at 37°C or 39 °C. Similar results were obtained using NIH3T3 fibroblasts which express transforming ΔXB-c-αb. or v-src oncogenes (data not shown). Taken together, these results indicate that temperature-induced sensitivity of wt BCR/abl kinase is only evident in hematopoietic cells and not in NIH3T3 fibroblasts which further underscores the fundamental differences in signal transduction between these cell types. We also examined the effect of temperature on the activity of an unrelated tyrosine kinase receptor, PDGFR, in NIH3T3 fibroblasts. As shown in Figure 2C, these cells had no change in activation of PDGF-R upon stimulation with PDGF β at 37°C or 39°C (Figure 2C). These results suggest that temperature dependent downregulation of BCR/abl kinase activity is restricted to Abl kinase expressed in hematopoietic cells.
EXAMPLE 3 - Effect of temperature on Abl and BCR/abl tyrosine kinase activity in vitro.
To determine the optimum temperature for BCR/abl tyrosine kinase in vitro, kinase activity of immunoprecipitated p210 BCR/abl was tested at different temperatures using tyrosine kinase substrate, raytide. Figure 3 shows the effect of temperature on p210 BCR/abl and recombinant purified Abl kinase.
In Figure 3A, p210 BCR/abl was immunoprecipitated from BaFp210 cell lysate and used for in vitro kinase activity. BCR/abl on Protein A/Sepharose beads was preincubated for 30 min at different temperatures ranging from 4 - 39°C. The kinase reactions were carried out at respective temperatures for additional 30 min, and kinase activity determined by the rate of 32P incorporation into raytide substrate. Data represents one of three independent experiments. As shown in Figure 3A, p210 BCR/abl in vitro kinase activity was maximal between 24°-28°C and reduced by 80% and 93% at 37°C and 39°C, respectively.
Figure 3B shows the effect of temperature on autophosphorylation of immunoprecipitated BCR/abl protein. After removing the clear supernatant as described in panel A, remaining samples were boiled in 1XSDS gel loading buffer, proteins fractionated on SDS-PAGE gels and dried gels were exposed for autoradiography. Data represents one of three independent experiments. Maximal autophosphorylation of BCR/abl occurred at 28°C (Fig 3B, lane 4) which was markedly reduced at 39°C to the levels observed at 4°C (Fig 3B, lane 7).
We also examined the effect of temperature on the activity of purified recombinant kinase domain of v-abl. Figure 3 shows the effect of temperature on the kinase activity of purified recombinant Abl. The temperature effect on purified Abl kinase was determined using GST-Crk as substrate. Kinase reactions were carried out at indicated temperatures as described under panel A. Each reaction contained 2 μg of GST-Crk and 2 units of Abl kinase. Data represents one of three independent experiments. Abl kinase activity was similar to that of p210 BCR/abl and was almost undetectable at 39°C (Fig 3C). Time course of inhibition of kinase activity for recombinant Abl revealed that in vitro inhibition was rapid and kinase activity was lost within 5 min of preincubation at 39°C (data not shown) which is significantly faster than the effect of temperature on the loss of tyrosine phosphorylated substrates observed in vivo. These results indicate that increase in temperature downregulates - fc>- tyrosine kinase activity.
EXAMPLE 4- Expression and activity of specific protein tyrosine phosphatases in BCR/abl transformed hematopoietic cells.
The differences in the rate of Abl kinase inhibition in vivo and in vitro suggested that the delay in disappearance of phosphorylated substrates in vivo could reflect the expression or activation of protein tyrosine phosphatases. In hematopoietic cells which express ts-p210 BCR/abl gene product enhanced expression and activation of PTP- 1B was linked to BCR/abl tyrosine kinase activity. (LaMontagne KRJ, et al., MolCell Biol. 1998; 18: 2965-2975). Increased expression of SH-PTPl and SH-PTP2 have been also described in BCR/abl transformed hematopoietic cells. (Sattler M, et al., Oncogene 1997; 15: 2379-2384). To understand whether delay in dephosphorylation of cellular proteins in BaFp210 cells at 39°C was due to altered expression or activity of protein tyrosine phosphatases, we performed Western blot analysis and measured in vitro phosphatase activity under linear assay conditions.
Figure 4 shows that expression or activity of protein tyrosine phosphatases in hematopoietic cell specific is not altered by temperature shift. Figure 4 A shows the expression of FTP- IB, SH-PTPl and SH-PTP2 protein tyrosine phosphatases in BaFp210 cells at 39°C. BaF (lane 1) or BaFp210 cells were grown at 37°C (lane 2) and shifted to 39°C (lanes 3 - 8) for 24 h. At indicated time intervals, cells were harvested and 50 μg of WCL protein was fractionated on SDS-PAGE gel, transferred onto nitrocellulose membranes and probed with anti-PTPlB, SH-PTPl or SH-PTP2 antibodies. Expression of FTP1B, SH-PTPl and SH-FTP2 remained constant at 39°C (Figure 4A). The apparent decrease in protein levels of SH-PTPl was not reproduced in other experiments.
Figure 4 B shows the total cellular protein tyrosine phosphatase activity is not altered in BaFp210 cells grown at 39°C. Total FTP activity from equal quantities of lysate proteins ( 1 - 2 μg) from BaF or BaFp210 cells grown at indicated temperatures was determined using Abl kinase phosphorylated raytide as a phosphate donor. Total phosphatase activity in whole cell extracts from cells grown at different temperatures overnight also showed no measurable difference between 37°C and 39°C (Fig 4B).
Phosphatase activity in immunodepleted cell extracts was also measured using anti-FTPlB, SH-PTPl or SH-PTP2 antibodies. Although FTP IB, SH-PTPl and SH-FTP2 were effectively immunoprecipitated with respective antibodies, the tyrosine phosphatase activity in immunodepleted supernatant was not altered (data not shown). Taken together, these results suggests that the loss of tyrosine phosphorylated proteins in cells maintained at 39°C reflects a lack of BCR/abl tyrosine kinase activity rather than an increase in protein-tyro sine phosphatase activity.
EXAMPLE 5 -The effect of downregulation of BCR/ abl tyrosine kinase activity on PI 3 kinase activity and cell growth.
Activation of PI 3-kinase has been shown to correlate with tyrosine kinase activity and survival of BCR/abl transformed hematopoietic cell lines and CML cells (Jain SK, et al., Blood 1996; 88: 1542-1550; Jain SK, et al., Oncogene 1997; 14: 2217-2228; Skorski T, et al., Blood 1995; 86: 726-736) whereas expression of BCR/abl in NIH3T3 fibroblasts does not lead to accumulation of PI 3-kinase lipid products. (Varticovski L, et al., Nature 1989; 342: 699-702). The effect of temperature on PI 3-kinase activity was analyzed in cells shifted to 39°C and during their recovery at 37°C.
Figure 5 shows that the down-regulation of PI 3-kinase activity by temperature shift at 39°C does not correlate with proliferation of hematopoietic cells. Figure 5 A shows that temperature-induces a reversible downregulation of PI 3-kinase. BaFp210 cells grown at 39°C for 18 h (lane 1) were returned to 37°C for indicated time (lanes 2 - 5) or grown at 37°C for 18 h (lane 6). PI 3-kinase activity was measured in anti-phospho-tyrosine immunoprecipitates from cells grown at indicated temperatures using PI and PIP2 as substrates. Data represents one out of four independent experiments. Total cellular PI 3-kinase activity in BaFp210 cells grown at 39°C for 16 or more hours was decreased to the basal levels equal to the levels of quiescent BaF cells (data not shown). Anti-phospho-tyrosine immunoprecipitable PI 3-kinase was also decreased by >90% in cells cultured at 39°C (Fig 5A) which correlated with the decrease in tyrosine phosphorylated substrates. Recovery of tyrosine kinase activity by shifting cells back to 37°C resulted in a rapid reactivation of PI 3-kinase in anti- phospho-tyrosine immunoprecipitates (Fig 5A). These results are similar to those seen in ts-BaFp210 cells grown at permissive temperature which allows activation of tsp210 BCR/abl kinase. (Jain SK, et al., Blood 1996; 88: 1542- 1550).
Because incubation of primary leukemic cells or BCR/abl transformed BaF cells with PI 3-kinase inhibitors, such as Wortmannin, partially (30%) blocks proliferation, we examined whether temperature dependent downregulation of BCR/abl tyrosine kinase activity and subsequent decrease in PI 3-kinase activity has an effect on cell proliferation. Figure 5 B shows that downregulation of BCR/abl activity by temperature does not affect cell growth or IL-3 independence. BaF and BaFp210 cells were cultured at 5 x 104 cells/ml at indicated temperatures (open circles: 37°C, closed circles: 39°C). Parental BaF cells were grown in the presence of 20% conditioned media from WEHI-3B cells. Number of live and dead cells were counted every 24 h by trypan blue exclusion. Data represents one out of six independent experiments.
Although tyrosine kinase activity could not be detected in cells grown at 39°C, there was no decrease in proliferation or morphological differences detected in BaFp210 cells grown at 39°C (Figure 5B). Addition of IL-3 also did not have an effect on cell growth at either temperature (data not shown). Similar results were obtained using BaF cells transformed by pi 90 and pl90Y177F BCR/abl (data not shown). These data suggest that the decrease in tyrosine kinase activity and shut-down of signaling pathways which require PI 3-kinase and GRB2 are insufficient to induce cell death of BCR/abl transformed hematopoietic cell lines. This is consistent with previous reports of secondary mutations in these cells which enable the cells to survive without BCR/abl activity, unlike cells in vivo. See e.g., Klucher, K.M. et al., Blood, 1998, 9:3927-3934.
EXAMPLE 6- Treatment of bone marrow or stem cells with ex vivo hyperthermic exposure.
The bone marrow of a patient having CML is removed by known methods in the art. The marrow is removed at the time of remission prior to treatment with chemotherapy or radiation therapy. The bone marrow is subjected to a temperature of 39°C, for about 16 to 24 hours, to kill any residual tumor cells. To increase the number of cells killed, an anti-CML agent, such as Interferon or abl kinase inhibitors, is added to the bone marrow. The anti-CML agent may be added prior to, during or after, hyperthermic treatment. The bone marrow is checked before and after using methods known in the art, such as RT-PCR, FISH, for the absence of tumor cells or for the presence of the Philadelphia chromosome. The tumor free bone marrow is then transplanted back into the patient, according to known methods.
In another example in which a donor's bone marrow is used, it would follow the above protocol.
EXAMPLE 7 - Methods of screening compounds for use as anti-CML agents in conjunction with hyperthermia.
An animal, e.g., mouse or rat, manifesting CML is anesthetized according to known methods. (See e.g., Daley, G.Q., et al., Science, 1990, 247:4944, 824-30; Daley, G.Q., et al., Proc. Natl. Acad. Sci., 1991, 88:24, 11335-8; Ilaria, R.L., et al., Blood, 1995, 86: 10, 3897-904). The mouse is subjected to increased external temperatures by any one of a number of methods such as high temperature hydrotherapy with immersion, hot warming blankets and insulation, forms of radiant energy and exracorporeal circulation. For example, the mouse is subjected to radiant heat. To prevent heat losses from the evaporation of perspiration, which would slow heating, the air surrounding the mouse is humidified. The animal is monitored to evaluate body temperatures and heart rate. The mouse is subjected to elevated temperatures until the desired temperature is reached. At this time the animal is removed and covered with blankets. While covered, the higher body temperature is maintained for the period of time specified in each treatment plan. At the conclusion of the treatment, the heat-retaining blankets are removed and body cooling begins.
The compound to be tested for anti-CML activity is administered prior to, during or after WBH. The compound to be tested is prepared in a suitable pharmacological carrier, such as normal saline, in an amount appropriate for the particular test mammal. Preferably, the compound is injected intravenously or intraperitoneally into the mammal. The effectiveness of the compound as an anti-CML agent is determined by methods known in the art.
The invention has been described in detail with particular references to the preferred embodiments thereof. However, it will be appreciated that modifications and improvements within the spirit and scope of this invention may be made by those skilled in the art upon considering the present disclosure.
The references cited herein are incorporated by reference.

Claims

We claim:
1. A method for treating a disease expressing the BCR/abl gene comprising subjecting a patient having the disease to whole-body hyperthermia (WBH), wherein the body temperature of the patient is raised to a temperature of at least about 38°C.
2. The method according to claim 1 , wherein the disease is myelogenous leukemia.
3. The method according to claim 1, wherein the disease is chronic myelogenous leukemia (CML).
4. The method according to claim 1 , wherein the body temperature is raised for at least about 2 hours.
5. The method according to claim 1 , wherein the whole body hyperthermia is repeated at periodic intervals.
6. The method according to claim 1, further comprising administering at least one anti-tumor agent to the patient prior to, during, or after the patient has undergone whole body hyperthermia.
7. The method according to claim 6, wherein the anti-tumor agent comprises a conventional therapy of chemotherapy or a biologic response modifier.
8. The method according to claim 7, wherein the biologic response modifier comprises interferon, abl kinase inhibitors or other inhibitors of BCR/abl downstream targets.
9. A method of reducing tumor cells in a stem cell preparation prior to transplantation into a patient suffering from myelogenous leukemia, comprising subjecting the bone marrow to a temperature above 37°C.
10. The method according to claim 9, wherein the stem cell preparation comprises bone marrow or stem cells collected from peripheral blood.
1 1. The method according to claim 9, wherein the temperature is raised within a range of about 38°C to about 42°C, preferably about 39°C.
12. The method according to claim 8, further comprising administering at least one anti-tumor agent to the stem cell preparation prior to, during, or after the heat treatment.
13. A method for treating chronic myelogenous leukemia (CML) comprising subjecting a patient having CML to whole-body hyperthermia (WBH) , wherein the body temperature of the patient is raised to a temperature of at least about 39°C.
14. A method of screening compounds for treating myelogenous leukemia comprising:
(a) subjecting an animal having myelogenous leukemia to whole-body hyperthermia (WBH) to raise the body temperature of the animal above normal,
(b) administering at least one compound suspected of having anti- tumor properties to the animal prior to, during, or after the animal has undergone whole body hyperthermia and
(c) determining the effectiveness of the compound.
15. A method of downregulating abl kinase activity of BCR/abl and v-abl transformed hematopoietic cells comprising subjecting the cells to temperatures above 37°C.
16. A method for treating chronic myelogenous leukemia (CML) comprising downregulating abl kinase activity by subjecting a patient having CML to whole-body hyperthermia (WBH), wherein the body temperature of the patient is raised to a temperature of at least about 39°C.
PCT/US1999/027392 1998-11-19 1999-11-19 Treatment of myelogenous leukemia WO2000028813A1 (en)

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