cell lines expressing bcr-abl synergy between imatinib and

doi:10.1182/blood-2004-10-3864Prepublished online December 16, 2004;

Jing Jin Gu, Lalaine Santiago and Beverly S Mitchell cell lines expressing Bcr-AblSynergy between imatinib and mycophenolic acid in inducing apoptosis in

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1

Synergy between Imatinib and Mycophenolic Acid in Inducing Apoptosis in Cell Lines Expressing Bcr-Abl

(Running head) Enhanced Imatinib cytotoxicity with GTP depletion.

Jing Jin Gu, Lalaine Santiago and Beverly S. Mitchell

Lineberger Comprehensive Cancer Center, Departments of Pharmacology and Medicine,

University of North Carolina at Chapel Hill, Chapel Hill, NC.

This study was supported by National Institutes of Health Grant CA 64193 to BSM

Corresponding author: Beverly S. Mitchell, MD, Departments of Pharmacology and

Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina,

Chapel Hill, NC27599-7295. Telephone: 919-966-4340. Fax: 919-966-8212. Email:

[email protected]

Total text word count: 4772 Abstract word count: 196 Scientific categories: Neoplasia

Blood First Edition Paper, prepublished online December 16, 2004; DOI 10.1182/blood-2004-10-3864

Copyright © 2004 American Society of Hematology


mailto:[email protected]



2

Abstract

Bcr-Abl tyrosine kinase activity initiates a number of intracellular signaling cascades that

result in leukemogenesis. Imatinib mesylate, a specific Bcr-Abl tyrosine kinase inhibitor,

has been highly successful in the treatment of chronic myelogenous leukemia. However,

the emergence of Imatinib resistance and the incomplete molecular response of a

significant number of patients receiving this therapy have led to a search for

combinations of drugs that will enhance the efficacy of Imatinib. We have demonstrated

that Mycophenolic Acid (MPA), a specific inosine monophosphate dehydrogenase

(IMPDH) inhibitor that results in depletion of intracellular guanine nucleotides, is

synergistic with Imatinib in inducing apoptosis in Bcr-Abl expressing cell lines. Studies

of signaling pathways downstream of Bcr-Abl demonstrated that the addition of MPA to

Imatinib reduced the phosphorylation of both Stat5 and Lyn, a Src kinase family member.

The phosphorylation of S6 ribosomal protein was also greatly reduced. These results

demonstrate that inhibitors of guanine nucleotide biosynthesis may synergize with

Imatinib in reducing the levels of minimal residual disease in CML and lay the

foundation for clinical trials in which IMPDH inhibitors are added to Imatinib in patients

who have suboptimal molecular responses to single agent therapy or who have

progressive disease.




3

Introduction Bcr-Abl, a chimeric oncogene, is formed by a reciprocal translocation between

chromosomes 9 and 22 (Philadelphia chromosome, Ph1), that places the c-ABL gene

under the transcriptional control of the BCR gene 1,2, resulting in the constitutive

activation of c-Abl tyrosine kinase 3,4. Depending on the breakpoint site in the Bcr gene,

two variants of the Bcr-Abl fusion proteins are generated 5. The p210 Bcr-Abl is present

in virtually all cases of chronic myelogenous leukemia (CML), while the p185 product is

present in approximately 20% of acute lymphoblastic leukemias 6. The Bcr-Abl tyrosine

kinase activates a number of downstream cell signaling pathways resulting in growth

factor-independent cell proliferation, altered cell adhesion and resistance to apoptosis

induced by other chemotherapeutic agents 7. The recently developed inhibitor of Bcr-Abl

kinase activity, Imatinib mesylate, induces a high percentage of complete hematologic

responses and partial or complete cytogenetic remissions in chronic phase CML 8,9.

Imatinib has high affinity for Bcr-Abl tyrosine kinase 10 and inhibits its activity by

binding to its ATP-binding pocket 11. Despite a high degree of selectivity, approximately

60% of CML patients retain the Ph1 chromosome after 18 months on Imatinib treatment 12 and more than 90% remain positive for Bcr-Abl transcripts, as measured by the

quantitative real-time polymerase chain reaction assay 13. Residual Bcr-Abl positivity is

clearly linked to risk for disease progression and the development of blast crisis.

Resistance to Imatinib eventually develops in the majority of patients treated in the blast

crisis stage of the disease, primarily due to the emergence of point mutations in or

amplification of the Bcr-Abl gene 14. The emergence of Imatinib resistance creates a

strong incentive to find non-toxic approaches to enhance its efficacy, including

combining Imatinib with a drug that enhances its cytotoxicity.

Inosine monophosphate dehydrogenase (IMPDH; EC1.1.1.205) is an essential,

rate-limiting enzyme for the generation of guanine nucleotides through the de novo

synthesis pathway. It catalyzes the NAD-dependent conversion of inosine

monophosphate to xanthine monophosphate, which is subsequently converted to

guanosine monophosphate (GMP) by GMP synthase. The only alternative pathway for

guanine nucleotide biosynthesis is through the salvage of guanine to GMP by

hypoxanthine-guanine phosphoribosyltransferase. IMPDH enzyme activity is composed




4

of the activities of two separate but very closely related IMPDH isoenzymes, termed type

I and type II, that share 84% amino acid identity 15. Expression of IMPDH, particularly

the type II enzyme, is significantly upregulated in many tumor cells, including leukemia

cells 16-19, presumably to meet a high demand for guanine nucleotides during cell

proliferation. Reduction of guanine nucleotides by inhibiting IMPDH activity effectively

induces cell cycle arrest in late G1 phase in lymphocytes 20,21 and results in differentiation 22-24 or apoptosis 25-27 in cultured cell lines depending on the cell type. The fact that these

effects on cell growth and differentiation are completely reversed by the addition of

guanine or guanosine to the medium demonstrates that the effects of IMPDH inhibitors

are specifically due to guanine nucleotide depletion. Inhibitors of IMPDH have been

used clinically as immunosuppressive agents 28-30 and have also been used to induce

hematologic responses in patients with CML in blast crisis 31,32 and in Bcr-Abl positive

AML patients33. However, the reduction in blast count is temporary and other studies

have suggested that IMPDH inhibitors may be better used in combination with other

chemotherapeutic drugs 34,35.

In the present study, we have demonstrated that a specific IMPDH inhibitor,

Mycophenolic Acid (MPA), can significantly enhance Imatinib cytotoxicity in Bcr-Abl

positive cells. We have also investigated the possible Bcr-Abl signaling pathways

affected by combining a specific inhibitor of Bcr-Abl tyrosine kinase with guanine

nucleotide depletion.




5

Materials and Methods

Recombinant retroviral infection The pSRMSVtkneo plasmid, containing the p185 form of Bcr-Abl was kindly provided

by Dr. C. Der (Lineberger Comprehensive Cancer Center, University of North Carolina

at Chapel Hill). Retrovirus was produced in 293T cells using pVpack system

(Stratagene, La Jolla, CA). Both 32D and FL5.12 cells were infected with control

retrovirus alone or retrovirus encoding the p185 Bcr-Abl fusion gene. Cells were

selected and maintained in the presence of 500 µg/ml G418. The p185 Bcr-Abl-

expressing cells were also selected for growth independent of IL-3.

Cell lines and reagents

The murine myeloid cell line 32D was maintained in RPMI-1640 medium supplemented

with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 10%

WEHI conditioned medium as a source of IL-3. The murine pre-B cell line FL5.12 (a

gift from C. B. Thompson, University of Pennsylvania, Philadelphia) was maintained in

RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 20 mM HEPES (N-

2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid), 55 µM 2-mercaptoethonal, and 10%

WEHI conditioned medium. Human leukemia cell lines K562, HL60 and U937

(originally obtained from ATCC, Rockville, MD) were maintained in RPMI-1640

medium supplemented with 10% heat-inactivated FBS. An Imatinib-resistant K562

derivative cell line K562R and LAMA84 cells were generously provided by Dr. S. Grant

(Commonwealth University, Richmond, Virginia). LAMA84 cells were maintained in

RPMI-1640 medium (without phenol-red) supplemented with 0.1 mM MEM non-

essential amino acid (Gibco, Caraday, CA), 2 mM L-glutamine (Gibco) and 1 mM MEM

sodium pyruvate (Gibco). All cell cultures were maintained in the presence of 100 µg/ml

penicillin and streptomycin.

Mycophenolic acid purchased from Sigma (St Louis, MO) was dissolved in

ethanol to a final concentration of 10 mM. Imatinib obtained from Novartis Pharm

(Basel, Switzerland) was dissolved in DMSO to a final concentration of 10 mM. Both




6

drugs were stored in aliquots in –20oC. In all experiments, control samples received the

equivalent amount of solvent used for drugs. Antibodies against total and phospho-Stat5

(Tyr 694), phospho-Src (Tyr 416), total and phospho-S6 (Ser 235/236) were all

purchased from Cell Signaling Technology (Beverly, MA). The anti-Lyn antibody and

protein A-beads were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-

phosphotyrosine (4G10) antibody was obtained from Upstate USA, Inc (Charlottesville,

VA). MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) was

purchased from Sigma and dissolved in PBS to a final concentration of 5 mg/ml, stored

aliquots in -20 oC.

MTT assay

Cells were seeded at approximately 1 x 104 in a final volume of 100 µl in 96-well flat-

bottom microtiter plates (Corning Costar, Corning, NY) with or without various

concentrations of drugs. To exclude effects of drugs on the OD reading, the highest

concentration of drug was added to the medium without cells to serve as the background

control. Plates were incubated at 37oC in a 5% CO2 incubator for the indicated times. At

the end of incubation, 15 µl of MTT were added to each well, and plates were incubated

at 37oC for another 4 hrs, followed by the addition of 100 µl 10% sodium dodecyl sulfate

(SDS) solution in 0.01M hydrochloric acid. Plates were incubated overnight in 37oC and

absorbance was measured at 595 nm against a reference wavelength at 650 nm using a

MAXline Microplate Reader (Molecular Devices, Sunnyvale, CA). The mean of

quadruplicate for each dose was used to calculate the IC50 and for all the Combination

Index values.

Combination Index (CI) calculation

CI values were calculated using Biosoft CalcuSyn program (Ferguson, MO). The dose-

effect relationships analyzed using the median-effect equation was first obtained for each

single drug by its serial dilution. A CI value for each combination treatment was then

calculated based on the formula D1/Dx1 + D2/Dx2, where Dx1 and Dx2 are the doses of

drug1 and drug 2 alone required to produce x% effect, and D1 and D2 are the doses of




7

drug 1 and drug 2 in combination required to produce the same effect 36,37. CI was used

to express synergism (CI<1), additive effect (CI=1) or antagonism (CI >1).

Annexin V staining and cell cycle analysis

Apoptosis was measured using the Annexin V-FITC Apoptosis Detection Kit I (BD

Pharmingen, San Diego, CA), following the staining procedure provided by the

manufacturer. Briefly, 2 to 3 x 105 cells after single or combination drug treatment were

washed twice with cold PBS and resuspended in 100 µl of 1x binding buffer for Annexin

V and propidium iodide (PI) staining. For cell cycle analysis, approximately 5 x 105 cells

were harvested after drug treatment, washed once in cold PBS and fixed in 80% EtOH

and stored at 4oC overnight. For PI staining, cells were centrifuged at 1,300 x g for 5

min, washed once with PBS containing 0.2% BSA (BSA/PBS), and pellets were

resuspended in 500 µl BSA/PBS containing 100 µg RNaseA and 50 µg PI and incubated

at 37oC for 30 min. Both Annexin V stained and cell cycle PI stained cells were analyzed

by flow cytometry. Approximately 30,000 cells were collected using FACScan Flow

(Becton Dickinson Immunocytometry, Mountain View, CA).

DNA fragmentation ELISA assay

Mononucleosomes and oligonucleosomes in the cytoplasm of the apoptotic cells were

measured using a Cell Death Detection ELISA kit (Roche Diagnostic GmbH,

Indianapolis, IN) according to the manufacturer’s protocol. Lysate from approximately 1

x 103 cells was used for each well. Absorbance was measured at 405 nm against a

reference of 490 nm using a MAXline Microplate Reader (Molecular Devices). All the

experiments were performed in duplicate in ELISA assays.

Western immunobloting

To prepare cell lysates for Western immunoblot analysis, cell pellets were lysed in buffer

containing 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% Glycerol, 1% TritonX-100, 1

mM Na3VO4, 25 mM β-glycerophosphate, 1x protease inhibitor cocktail and 1x

phosphatase inhibitor cocktail 1 (Sigma). After centrifugation at 14,000 x g for 15 min,

protein concentrations were quantitated in duplicate by the Bradford method (Bio-Rad




8

Laboratories, Hercules, CA). Equal amounts of protein were separated on a SDS-

polyacrylamide gel electrophoresis (PAGE), and transferred to either polyvinylidene

difluoride Immobilon-P membranes (Millipore Corp. Bedford, MA) or Protran

nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The membranes were

blocked with 5% nonfat milk made in Tris-Buffer Saline containing 0.1% Tween 20

(TBST0.1) at room temperature for 1 hr, and then incubated with primary antibodies

diluted either in 5% BSA for phospho-protein detection or in blocking solution for other

antibodies at 4oC overnight. Dilutions of primary antibodies were made following the

manufacturer’s instructions. Following several washes in TBST0.1, membranes were

incubated with appropriate secondary antibodies (1:2000 to 1:5000 dilution, Amersham,

Co. Arlington Heights, IL) at room temperature for 1 hr. Detection was performed using

the ECL chemiluminescence system (Amersham).

Immunoprecipitation

Immunoprecipitation (IP) of Lyn was performed following the protocol described by

Danhauser-Riedl et al38. Briefly, 1 mg of total lysate was incubated with 5 µg of anti-

Lyn antibody. After rotating 3 hrs at 4oC, 100 µl of protein A-Beads (50% beads volume)

was added for an additional 2 hrs. Precipitated proteins were separated on 10% SDS-

PAGE and blotted with anti-phosphotyrosine antibody (4G10).




9

Results Effects of Imatinib and MPA on growth of Bcr-Abl transformed cell lines

Murine 32D myeloid and FL5.12 pre-B cells were infected with retrovirus carrying the

p185 Bcr-Abl fusion protein or an empty vector (as described in the Methods). Both cell

lines appropriately expressed the Bcr-Abl protein, as demonstrated by Western blots (data

not shown). Inhibition of cell growth by MPA or Imatinib was then analyzed using MTT

assays 37. As indicated in Table 1, the IC50 values for MPA are similar across cell lines

regardless of Bcr-Abl expression. Consistent with previous results39, 32D and FL5.12

cell lines expressing Bcr-Abl are highly sensitive to Imatinib with IC50 values of

approximately 0.6 µM, whereas vector control cells are completely resistant to Imatinib-

induced growth inhibition up to the highest concentration (8 µM) tested. Growth

inhibition data for these two drugs were also obtained for two human leukemia cell lines

expressing p210 Bcr-Abl, K562 and LAMA84, and two Bcr-Abl negative leukemic cell

lines, HL-60 and U937. The growth of all four-cell lines is inhibited by MPA at similar

concentrations, albeit at longer incubation times than for the murine cells. In contrast, the

two Bcr-Abl-negative cell lines are completely resistant to Imatinib concentrations up to

8 µM, whereas the K562 and LAMA84 cells are very sensitive to Imatinib with IC50

values of 0.5 and 0.1 µM respectively.

To study the possible synergy between Imatinib and MPA, approximately

equipotent concentrations of each drug were used. Combination index (CI) values were

calculated using CalcuSyn software (see Methods), where CI=1 indicates an additive

affect, CI<1 indicates synergy and CI>1 indicates antagonism calculated using a formula

initially proposed by Chou and Talalay 36. The two drugs were used in constant ratios in

most MTT assays to generate classical isobolograms. In some instances, non-constant

ratios were also tested to determine the effectiveness of a broader range of combinations

(data not shown). The CI values at ED50, ED75 and ED90 generated from classical

isobolograms for all four Bcr-Abl positive cell lines are summarized in Table 2.

Combined treatment with MPA and Imatinib demonstrates an additive (at about the

ED50) to synergistic (above the ED50) effect on growth inhibition in Bcr-Abl

expressing-32D and -FL5.12 cell lines. Combination treatment is clearly synergistic at

and above the ED50 in both human Bcr-Abl positive cell lines. Since the cell lines not




10

expressing Bcr-Abl are insensitive to Imatinib and data obtained for the combination are

similar to those obtained with MPA alone, the combination index calculation is not

applicable.

Effect of MPA and Imatinib combinations on the induction of apoptosis

To determine whether combined treatment with MPA and Imatinib also enhances

apoptosis, 32D-p185 and K562 cells were treated with various concentrations of MPA

and/or Imatinib followed by Annexin V staining and analyzed by flow cytometry. Each

drug alone at the doses used induced very little or no apoptosis; however, the

combination of the two greatly increased the percentage of Annexin V-positive cells in

both cell lines (Fig. 1). To determine the CI values for apoptosis, at least 4 doses of each

drug was used in every experiment to generate single drug curves. Highly synergistic

effects were observed for multiple combination treatments in both 32D-p185 and K562

cell lines (Table 3). Interestingly, the CI values for apoptosis demonstrate far more

synergy than do the cell growth inhibition experiments. This is most likely because each

drug alone can significantly inhibit growth but only induce very low levels of apoptosis at

the concentrations used. The reduction in live cells, as defined by both Annexin V and PI

negativity, for each combination treatment as compared to each drug alone was also

statistically significant (p<0.05) using a paired two-tailed Student t test analysis on three

independent experiments (data not shown).

Specificity of induction of apoptosis for Bcr-Abl-positive cells

To determine whether the induction of apoptosis by combined treatment with MPA and

Imatinib is specific for Bcr-Abl expressing cells, we treated vector control or Bcr-Abl-

expressing-32D and FL5.12 cells with MPA and/or Imatinib for 16 to 20 hrs and

fragmented DNA was analyzed using the Cell Death Detection ELISA method 27. The

level of apoptosis induced by the combination treatment in vector-control cells was

similar to that of MPA alone, whereas the combination markedly enhanced apoptosis in

Bcr-Abl expressing-cells (Fig. 2). Apoptosis was also evaluated by the size of the sub-

G1 peak obtained from cell cycle analysis. Bcr-Abl positive FL5.12 and LAMA84 cells

and Bcr-Abl negative HL-60 and U937 cells were analyzed by flow cytometry after




11

single and combination drug treatment (Fig. 3). In both FL5.12-p185 and LAMA cells,

MPA and Imatinib alone induced a small increase in the sub-G1 peak, whereas this peak

was markedly increased by the combination treatment. In HL-60 and U937 cells,

Imatinib (4uM) did not induce the apoptotic sub-G1 peak, nor did it enhance the MPA

effect.

Synergistic activity in K562 cells resistant to Imatinib

To determine whether MPA also enhance the response to Imatinib in cells that

demonstrate Imatinib resistance, we tested the effect of the combination on induction of

apoptosis using a K562 derivative cell line selected in the presence of Imatinib

(K562R40). As shown in Figure 4, K562R cells were significantly less sensitive to

Imatinib-induced apoptosis. K562 parental and K562R cells have similar sensitivity to

MPA treatment. The combination of MPA and Imatinib significantly enhanced the

induction of apoptosis in both K562 parental and K562R cells.

Effect of Imatinib and MPA on Stat5 phosphorylation

To study the possible mechanism(s) by which the combination of Imatinib and MPA

caused a synergistic effect on growth inhibition and apoptosis, we measured the

expression of several downstream effectors of Bcr-Abl signaling that have been

demonstrated to play important roles in Bcr-Abl transformation. The signal transducer

and activator of transcription (Stat) 5 was highly phosphorylated in 32D-p185, FL5.12-

p185 cells and in both K562 and LAMA84 human cell lines as compared to vector-

transfected 32D and FL5.12 parental cells and HL-60 and U937 cells (data not shown).

To determine whether MPA and/or Imatinib treatment decreases Stat5 phosphorylation,

we treated 32D-p185 and FL5.12-p185 cell lines with either drug alone or in combination

for 90 min. This time period is far shorter than the 4 to 8 hrs required for caspase 3

activation in these two cell lines (data not shown). Although Imatinib alone potently

inhibited Stat5 phosphorylation (p-Stat5), the combination of Imatinib and MPA further

reduced the level of p-Stat5 (Fig. 5A and B). Similar effects are also seen in K562 and

LAMA cells, although at much later time points (between 8 to 24 hrs; Fig. 5C and D).

The reduction of p-Stat5 levels by Imatinib alone at 24 h is less than the reduction at 3




12

and 8 hrs, indicating the effect of Imatinib is relatively short. However, the combination

of Imatinib and MPA maintained lower levels of p-Stat5 at 24hrs. This prolongation of

effect may have biologic significance in view of the 48 to 72 hrs it takes for the

combination to induce apoptosis in human cell lines. We have also demonstrated (data

not shown) that Mcl-1 and Bcl-xL, both downstream targets of p-Stat5, are significantly

reduced at these time points, underscoring the biologic consequences of reducing p-Stat5

levels41,42.

Effect of combination treatment on phospho-Jak2 and phospho-Src expression

We next examined upstream molecules that could phosphorylate Stat5. We failed to

identify significant amounts of constitutively activated Janus kinase (Jak) 2 protein in our

Bcr-Abl expressing cell lines, and did not detect any significant effect on phospho-Jak2

levels by either single or combination drug treatment (data not shown), suggesting that

Jak2 is not responsible for the effect on Stat5 phosphorylation. This result is also

consistent with the previous finding that inhibition of Jak2 does not affect Stat5 activity

in Bcr-Abl positive cells43.

Increasing evidence has indicated that Stat proteins can also be activated through

members of the Src gene family 44. In particular, two family members, Lyn and Hck, can

phosphorylate Stat5 45,46, and have been implicated in Bcr-Abl-mediated transformation 38,47. We have found that levels Src phosphorylation in 32D and FL5.12 cells were very

high independent of the expression of p185 Bcr-Abl, most likely due to the continuous

IL-3 stimulation required for the growth of vector-control cells. However, Src is more

highly phosphorylated in the human Bcr-Abl positive cell lines, K562 and LAMA84

cells, than in HL-60 and U937 cells (data not shown). Although neither drug by itself

significantly decreased p-Src levels, the combination dramatically reduced Src

phosphorylation in 32D-p185 and FL5.12-p185 cells after 90 min (Fig. 6A). Similar,

although less dramatic, reductions were observed after 24 hrs of combined treatment of

K562 and LAMA cells (Fig. 6A). The antibody used to detect p-Src (Y416) detects

several activated Src family members, including Lyn (Y396, p53/p56) and Hck (Y390,

p59). When the blots were probed with total Lyn, Hck or Src antibodies, two bands

detected by the Lyn antibody, p53 and p56, corresponded precisely to the two bands




13

detected by p-Src antibody. In contrast, Hck-specific antibody demonstrated a single

slightly higher band, the p59 protein, while Src-specific antibody also recognized a p60

protein. To confirm that the reduction in phosphorylation is that of Lyn, we performed

immunoprecipitation using anti-Lyn antibody followed by Western blot analysis with a

tyrosine phosphorylation-specific antibody (Fig. 6B). These results clearly demonstrated

that Lyn phosphorylation is reduced by the combination treatment.

Effect of combination treatment on mTOR pathway

It has been shown that the mammalian target of Rapamycin (mTOR) pathway is activated

in Bcr-Abl transformed cells 48, as indicated by hyper-phosphorylation of p70 S6 kinase

and S6 ribosomal proteins, as well as 4EBP1, resulting in enhanced protein translation

and cell growth. We have previously shown that the p70 S6 kinase phosphorylation is

downregulated by MPA treatment in 32D cells 27. We therefore examined the S6

phosphorylation level following MPA and/or Imatinib treatment in both murine and

human Bcr-Abl expressing cell lines. The level of phosphorylated S6 (p-S6) was

slightly reduced by either MPA or Imatinib treatment alone, but was markedly reduced

with combination of MPA and Imatinib (Fig. 7).




14

Discussion: Inhibition of the enzyme IMPDH and the consequent guanine nucleotide

depletion have previously been shown to reduce blast counts in patients with CML in

blast crisis31-33, but the effect was transient and the toxicity of the drug precluded further

investigation. Although inhibition of IMPDH has also been shown to result in the

differentiation of leukemic cell lines such as HL-60 and K-562 23,49 and to cause either

cell cycle arrest 20,21 or apoptosis 25-27 in other cell lines, the alterations in specific

signaling pathways that result in these effects have not been fully elucidated. In previous

work, we demonstrated that inhibition of the de novo synthesis of guanine nucleotides in

IL-3 dependent cell lines resulted in apoptosis, while simultaneously causing the early

down-regulation of both the MEK/Erk and the mTOR signaling pathways 27. These

results and the demonstration of synergy between MPA and Imatinib in Bcr-Abl

expressing cells led us to examine the effects of combined treatment on specific signaling

molecules that have been demonstrated to be important in Bcr-Abl mediated

transformation.

The Stat proteins are very important in Bcr-Abl-mediated oncogenesis 50,51.

These transcription factors are normally present in the cytoplasm of quiescent cells and

are phosphorylated in response to stimulation by cytokines or growth factors, resulting in

dimerization and nuclear translocation, and ultimately to the expression of specific target

genes 52. Stat5 is the major Stat protein activated by Bcr-Abl in cell lines and in

hematopoietic cells derived from patients with CML 43,53. Although an increase in Stat5

phosphorylation alone, using a dominant-active Stat5, is not sufficient to transform

primary murine bone marrow cells, expression of a dominant negative Stat5 reduces the

efficiency of Bcr-Abl-mediated transformation and impairs leukemogenesis 54. It has

also been shown that the anti-apoptotic effect of Bcr-Abl is dependent upon Stat5

signaling to activate the Bcl-2 family members, Bcl-xL and Mcl-1 55,56. We have

demonstrated that MPA treatment significantly enhanced Imatinib’s ability to decrease

Stat5 phosphorylation and also reduced Mcl-1 and Bcl-xL expression. This effect was

also enhanced at later time points in human cell lines, suggesting that the inhibitory effect

of Imatinib can be extended by treatment with MPA.




15

The Src family of tyrosine kinases are known to phosphorylate Stat proteins 44.

The two Src family members, Lyn and Hck, are highly activated in Bcr-Abl expressing

cells 38 as a results of direct physical interaction with Bcr-Abl 57,58. Expression of a

kinase-defective mutant of Hck significantly down-regulated p-Stat5 46 and prevented

transformation by Bcr-Abl 47, while inhibition of Lyn kinase expression using antisense

oligonucleotides also significantly reduced leukemia cell growth 59,60. Our results have

demonstrated that the combination of MPA and Imatinib markedly reduces Lyn

phosphorylation. Since overexpression of activated Lyn has been documented in later

stages of CML and in Bcr-Abl expressing cells that have developed resistant to Imatinib 60, the additive effect of MPA on reducing p-Lyn may well be pivotal to its synergistic

effect.

Although the PI3 kinase pathway is believed to be important for Bcr-Abl

transformation and leukemia cell proliferation 61,62, we failed to observe any change in

phospho-Akt level at either Thr 308 and Ser 473 sites following either MPA or Imatinib

alone or in combination. However, we did observe a dramatic down-regulation of the

phosphorylated S6 ribosomal protein, a direct target of the mTOR/S6K1 pathway. It has

been demonstrated previously that a Bcr-Abl mutant that failed to activate PI3K/Akt can

still activate S6K1 62, indicating that the mTOR pathway may be activated independent of

the PI3K/Akt pathway. The importance of the mTOR pathway in CML has become

increasingly evident. This pathway acts as a sensor for the deprivation of essential

nutrients such as amino acids, sources of nitrogen or ATP 63,64 and regulates the

translation of a specific subset of mRNAs that contain polypyrimidine tracts at their 5’

ends 65. Both ribosomal protein S6, a substrate of S6K1, and 4EBP1 are constitutively

phosphorylated in CML cells 48. A very recent paper has documented synergy between

the mTOR inhibitor, Rapamycin, and Imatinib in Bcr-Abl expressing cell lines, as well as

synergy of Rapamycin with PKC412, an inhibitor of Flt-3 tyrosine kinase, in cell lines

overexpressing Flt-3 66. The addition of a MEK inhibitor further potentiated the

cytotoxicity. Clearly, pharmacological agents that inhibit multiple signaling pathways

including mTOR have the potential to be more effective than single agents in inducing

apoptosis in malignant cell lines.




16

Down-regulation of several signaling pathways caused by GTP depletion may

enhance the effects of several other more specifically targeted chemotherapeutic agents.

Our hypothesis is that guanine nucleotide depletion has a central sensing mechanism with

multiple downstream effectors, ultimately resulting in growth arrest or apoptosis.

Although this molecular sensor has not been definitively identified, we are exploring

several small GTP binding proteins as candidates. One, the Rheb GTPase, was initially

isolated as a Ras homolog enriched in brain 67 and directly activates the mTOR pathway 68. Transient expression of a wild-type Rheb induces a dramatic increase in S6K

phosphorylation and mobility shift of 4EBP1, and this high level of activity is consistent

with its being predominantly GTP bound68. A dominant negative Rheb, defective in GTP

binding, was found to block nutrient and serum-induced p70 S6K activation 69. We are

currently testing the hypothesis that a reduction of intracellular GTP resulting from

inhibition of de novo guanine nucleotide synthesis might decrease the level of GTP-

bound Rheb, thus inhibiting the mTOR/S6K1 pathway.

Our data suggest that the IMPDH inhibitors may constitute a major addition to the

armamentarium of drugs that target oncogenic receptor tyrosine kinases and we are

implementing a clinical protocol to test the combination of Imatinib and MPA in patients

with persistent positivity for the Bcr-Abl transcript on Imatinib alone after 12 months.

Our results demonstrating enhanced toxicity of the combination over either agent alone in

cells that are partially resistant to Imatinib on the basis of Lyn overexpression 40

constitutes preliminary evidence that partial resistance to Imatinib may be overcome by

this approach. Although we have attempted to test this hypothesis in the leukemic cells

from two cases of CML in blast crisis, the high degree of baseline apoptosis resulting

from incubation of these cells ex vivo made interpretation of the results impossible.

Guanine nucleotides can be synthesized directly through the salvage of guanosine

in vivo, thereby compensating for IMPDH inhibition. However, it has been

demonstrated that Imatinib inhibits nucleoside uptake through selective inhibition of

nucleoside transporters 70 . We have confirmed that Imatinib treatment blocked the

nucleoside transporter in a dose-dependent manner in leukemic blast cells from a CML

patient (data not shown). Addition of guanosine to the culture medium did not reverse

the synergistic effects of MPA and Imatinib (data not shown). This observation enhances




17

the probability that this combination will be effective in patients, since Imatinib will

inhibit purine salvage. The proposed clinical trial will be the optimal approach to

definitively test our hypothesis and will incorporate the specific molecular endpoint of a

greater than 1 log reduction in the Bcr-Abl transcript six months after the addition of

MPA.




18

Acknowledgment We thank Lee Graves for reading and critiquing the manuscript.




19

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760




27

Table 1: Effect of MPA or Imatinib on cell growth in Bcr-Abl positive and negative cell lines

Cell Lines Description IncubationTime (hrs)

IC50 MPA (µM)

IC50 Imatinib (µM)

32D-Vector Murine myeloid 24 0.46 (±0.16) >8

32D-p185 Murine myeloid (p185 Bcr-Abl) 24 0.56 (±0.14) 0.56 (±0.08)

FL5.12-Vector Murine pre-B 24 0.4 (±0.16) >8

FL5.12-p185 Murine pre-B (p185 Bcr-Abl) 24 0.8 (±0.14) 0.68 (±0.2)

K562 Human CML (p210 Bcr-Abl) 72 0.41 (±0.17) 0.53 (±0.07)

LAMA84 Human CML (p210 Bcr-Abl) 48 0.47 (±0.03) 0.089 (±0.01)

HL-60 Human

Promyelocytic Leukemia

48 1.0 (±0.2) >8

U937 Human Monocytic Leukemia 48 0.65 (±0.37) >8

IC50 values were obtained using the MTT assays. Values represent the mean ± S.D. for at least three experiments.




28

Table 2: Combination Index values for growth inhibition

CI Values Cell Lines MPA: Imatinib

ED50 ED75 ED90

32D-p185 2:1 0.94 (±0.2) 0.64 (±0.03) 0.50 (±0.07)

FL5.12-p185 2:1 0.88 (±0.07) 0.77 (±0.06) 0.76 (±0.03)

K562 4:1 0.63 (±0.1) 0.27 (±0.02) 0.58 (±0.1)

LAMA84 8:1 0.79 (±0.1) 0.53 (±0.09) 0.58 (±0.05)

Combination index values were calculated from the MTT assays. Constant ratios of two drugs were used to obtain the isobologram ED values. Data represent the mean ± S.D. of three independent experiments.




29

Table 3: Combination Index values for induction of apoptosis

Cell Lines Drugs (µM) CI Description

32D-p185 MPA (1) + Imatinib (0.5) 0.47 (± 0.13) Synergism

MPA (1) + Imatinib (1) 0.28 (±0.07) Strong Synergism

MPA (2) + Imatinib (0.5) 0.34 (± 0.04) Synergism

MPA (2) + Imatinib (1) 0.21(± 0.09) Strong Synergism

K562 MPA (1) + Imatinib (0.25) 0.04 (± 0.04) Very strong Synergism

MPA (2) + Imatinib (0.5) 0.04 (± 0.05) Very strong Synergism

MPA (4) + Imatinib (1) 0.06 (± 0.08) Very strong Synergism

CI values were obtained by Annexin V staining analyzed by flow cytometry. Data represent the mean ± S.D. of three independent experiments.




30

Figure Legends

Figure 1: Effect of combined treatment with Imatinib and MPA on the induction of

apoptosis. 32D-p185 (A) and K562 (B) cells were treated with MPA and/or Imatinib at

the doses indicated for 24 and 72 hrs, respectively. Apoptosis was measured by Annexin

V and PI staining. The percentage of Annexin V-negative and -positive cells are

indicated on each histogram. The Annexin V positive peak shown in the histogram

includes both the AnnexinV-positive, PI-negative and the Annexin V-positive, PI-

positive populations. At least three experiments with multiple drug combinations were

performed on each cell line. One representative experiment at a selected combination of

doses is shown.

Figure 2: Effect of combination treatment on the induction of DNA fragmentation in

Bcr-Abl-expressing cells. Both 32D (A) and FL5.12 (B) vector control cells (Bars 1-4)

or p185-expressing cells (Bars 5-8) were either not treated (white bars) or treated with

MPA alone (Black bars; 1µM for 32D, 0.5 µM for FL5.12) or Imatinib alone (Gray bars;

1µM for 32D, 0.5 µM for FL5.12 cells) or the combination of MPA and Imatinib (striped

bars) for 16-20 hrs and apoptosis was measured by the Cell Death Detection ELISA

method. Data represent mean ± S.D. of three independent experiments. ELISA assays

were performed in duplicate.

Figure 3: Induction of apoptosis in Bcr-Abl positive and negative cell lines using cell

cycle analysis. FL5.12-p185, LAMA84, HL-60 and U937 cells were treated with the

indicated concentrations of MPA and/or Imatinib for 24 hrs (FL5.12-p185) and 48 hrs (all

other cell lines). The percent of sub-G1 (apoptotic) cells are shown on each histogram.

At least three experiments with multiple drug combinations were performed on each cell

line. One representative experiment at one selected dose combination is shown.

Figure 4: Effect of combination treatment on Imatinib-resistant cells. K562 and

K562R cells were treated with Imatinib at the indicated doses for 72 hrs with or without

MPA. Apoptosis was measured by Annexin V and PI staining. Imatinib treatment alone




31

is indicated by the closed symbols. The controls in the absence of drug have 10.2 % ±

2.2 and 10.9 % ± 2.7 Annexin V/PI positive cells for K562 and K562R, respectively.

Imatinib plus MPA (2 µM) is indicated by the open symbols. MPA alone induced 22.5

% ± 0.6 and 26.3 % ± 2.3 apoptosis for K562 and K562R, respectively. Data represent

the mean ± S.D. of three independent experiments.

Figure 5: Effect of combination treatment on Stat5 phosphorylation. 32D-p185 (A),

FL5.12-p185 (B), K562 (C) and LAMA84 (D) cells were treated with indicated

concentrations of MPA and/or Imatinib for 90 min (for 32D- and FL5.12-p185) or the

indicated times for K562 and LAMA84 cells. 30 µg of total cell lysate were loaded in

each lane and Western blot analysis was performed using an antibody specific for

phospho-Stat5 (p-Stat5). Duplicate gels were probed with antibody specific for total

Stat5 (t-Stat5). 32D-p185 and FL5.12-p185 results are the representative of 4 and 3

independent experiments, respectively. K562 and LAMA84 results are representative of

two independent experiments; treatment for 24 hrs has been repeated three times.

Figure 6: Effect of combination treatment on Src and Lyn phosphorylation. (A).

32D-p185, FL5.12-p185, K562 and LAMA84 cells were treated with the indicated

concentrations of MPA and/or Imatinib for 90 min (for 32D- and FL5.12-p185 cells) and

24 hrs (for K562 and LAMA84 cells). Total cell lysate (30 µg) was used in each lane.

Western blots were first probed with anti-phospho-Src antibody. Membranes were

washed and re-probed with an antibody to detect total levels of Lyn (t-Lyn). This result

is representative of three independent experiments. (B). 32D-p185 cells were treated

with single or the combination of drugs as indicated for 3 hrs. Lysate was

immunoprecipitated with anti-Lyn antibody and blotted with an antibody (4G10) specific

for tyrosine phosphorylated proteins (p-tyr). Lysate precipitated with protein A-beads in

the absence of Lyn antibody (IP control) and total lysate are shown in the last two lanes.

Figure 7: Effect of combination treatment on S6 phosphorylation. 32D-p185, FL5.12-

p185, LAMA84 and K562 cells were treated with the indicated concentrations of MPA

and/or Imatinib for 3 hrs (for 32D- and FL5.12-p185 cells) and 24 hrs (for LAMA84 and




32

K562 cells). Western blots were probed with antibody specific for phospho-S6 ribosomal

protein (p-S6). Duplicate gels were probed with antibody specific for total S6 (t-S6).

This is representative of three independent experiments.




33

Figure 1

Annexin V

Cel

lCou

nts

Control

Control

Imatinib 1 µM

Imatinib 0.5 µM

MPA 2 µM

MPA 2 µM

Combined

Combined

86 14 74 26 66 34 28 72

72 28 82 18 73 27 37 63

A.

B.

32D-p185

K562




34

Figure 2

A. 32D-vector/p185

Frag

men

ted

DN

A(O

D40

5-49

0nm

)

0.0

0.5

1.0

1.5

2.0

2.5

1 2 3 4 5 6 7 8

DMSO/EtOH ControlMPAImatinibMPA + Imatinib

B. FL5.12-Vect-p185

Frag

men

ted

DN

A(O

D40

5-49

0nm

)

0.0

0.5

1.0

1.5

2.0

2.5

1 2 3 4 5 6 7 8




35

Figure 3

DNA area

Cel

lCou

nts

FL5.12-p185

Control Imatinib MPA Combined0.5 µM 0.5 µM

0.125 µM 1 µM

4 µM 1 µM

1 µM4 µM

4 23 12 71

9 17 6 54

0.7 0.7 43 38

4 2 14 15

LAMA

HL-60

U937




36

Figure 4

Imatinib (µM)

Perc

ento

fAnn

exin

V/P

IPos

itice

Cel

ls

0

20

40

60

80

100

K562 Imatinib aloneK562 Imatinib + MPAK562R Imatinib aloneK562R Imatinib + MPA

0 0.25 0.5 1 2




37

Figure 5

Imatinib (µM)MPA (µM)

3h 8h 24h


3h 8h 24h

C.

p-Stat5

p-Stat5


A.

p-Stat5


B.

p-Stat5

- 1 - - - 1 1 1- - 0.5 1 2 0.5 1 2

- 0.5 - - - 0.5 0.5 0.5- - 1 2 4 1 2 4

- - 0.5 0.5 - 0.5 0.5 - 0.5 0.5- 2 - 2 2 - 2 2 - 2

- - 0.125 0.125 - 0.125 0.125 - 0.125 0.125- 2 - 2 2 - 2 2 - 2

t-Stat5

t-Stat5

t-Stat5

t-Stat5

D.




38

Figure 6

- - 1 1 - - 1 1- 2 - 2 - 2 - 2


32D-p185 FL5.12-p185

p-Src

t-Lyn

- - 0.5 0.5 - - 0.125 0.125- 2 - 2 - 2 - 2

K562 LAMA84

p-Src

t-Lyn


A.

B.

Imatinib (µM)MPA (µM) - - 2 2 -

- 1 - 1 -

Lyn IP

IPCon

trol

Lysate

p-tyr




39

Figure 7



- - 1 1 - - 0.5 0.5- 2 - 2 - 2 - 2

- - 0.125 0.125 - - 0.5 0.5- 2 - 2 - 2 - 2

32D-p185 FL-p185

LAMA84 K562

p-S6

p-S6

t-S6

t-S6




cell lines expressing bcr-abl synergy between imatinib and

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