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Cancer Therapy: Preclinical |
Authors' Affiliations: 1 Division of Hematology and Medical Oncology, Oregon Health and Sciences University, Portland, Oregon; 2 III. Medizinische Universitätsklinik, Fakultät für klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany; 3 Cell Therapeutics, Inc., Seattle, Washington; 4 Imperial College, Hammersmith Hospital, London, United Kingdom; and 5 Howard Hughes Medical Institute, Chicago, IL
Requests for reprints: Michael W. Deininger, Division of Hematology and Medical Oncology, Oregon Health and Science University Cancer Institute, Mail Code L592, 3181 Sam Jackson Park Road, Portland, OR 97239. Phone: 503-494-1603; Fax: 503-494-3366; E-mail: deinige{at}ohsu.edu.
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Abstract |
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Experimental Design: In this study, we investigated the effects of CT32228, a specific LPAAT-ß inhibitor, on BCR-ABL-transformed cell lines and primary cells from patients with chronic myelogenous leukemia.
Results: CT32228 had antiproliferative activity against BCR-ABL-positive cell lines in the nanomolar dose range, evidenced by cell cycle arrest in G2-M and induction of apoptosis. Treatment of K562 cells with CT32228 led to inhibition of extracellular signal-regulated kinase 1/2 phosphorylation, consistent with inhibition of mitogen-activated protein kinase signaling. Importantly, CT32228 was highly active in cell lines resistant to the Bcr-Abl kinase inhibitor imatinib. Combination of CT32228 with imatinib produced additive inhibition of proliferation in cell lines with residual sensitivity toward imatinib. In short-term cultures in the absence of growth factors, CT32228 preferentially inhibited the growth of granulocyte-macrophage colony-forming units from chronic myelogenous leukemia patients compared with healthy controls.
Conclusion: These data establish LPAAT-ß as a potential drug target for the treatment of BCR-ABL-positive leukemias.
CML can effectively be treated with the Bcr-Abl-directed kinase inhibitor imatinib (Gleevec), with excellent response rates in early-stage CML (10). However, patients in advanced CML relapse with high frequency (11). Moreover, imatinib fails to induce a complete cytogenetic response in 
15% of patients in chronic phase CML, and only a minority of patients achieve complete molecular remission as detected by highly sensitive PCR assays, a situation referred to as disease persistence at the molecular level (12). Several mechanisms causing primary resistance have been proposed, including quiescence of leukemic progenitor cells (13), drug efflux (14), mutations in the imatinib-binding kinase domain (15), or the activation of alternative pathways, including MAPK (16). It is thought that the latter may be compensating for inactivation of the Bcr-Abl kinase.
CT32228, an aryldiaminotriazine, is a small-molecule isoenzyme-specific inhibitor of LPAAT-ß. The antiproliferative IC50 in a screen of multiple solid tumor cell lines was in the low nanomolar range (17). CT32228 and related compounds are highly antiproliferative and proapoptotic in multiple myeloma cells (18). Biochemical studies showed inhibition of Ras/Raf/MAPK and phosphatidylinositol 3-kinase/Akt pathways leading to cell cycle arrest and apoptosis (17). However, little is known about the antileukemic potency of LPAAT-ß inhibitors in leukemia.
Given the involvement of MAPK signaling in the pathogenesis of CML and its potential role in imatinib resistance, we decided to evaluate the effects of CT32228 in inhibiting LPAAT-ß activity with CT32228 in BCR-ABL-positive cell lines and primary CML cells. Moreover, we explored the activity of this small molecule in cell lines resistant to imatinib.
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Materials and Methods |
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Imatinib was prepared as a 10 mmol/L stock solution in sterile PBS and stored at –20°C. CT32228 was prepared as a 10 mmol/L stock solution in DMSO and stored at –80°C.
Colony-forming assays. Colony-forming assays in the presence of 50 ng/mL recombinant human stem cell factor, 10 ng/mL recombinant human GM-CSF, and 10 ng/mL recombinant human IL-3 with 3 units/mL recombinant human erythropoietin [for culture of blast-forming unit erythroid (BFU-E)] or without erythropoietin [for culture of granulocyte-macrophage colony-forming unit (CFU-GM)] were done as described previously (26). Normal bone marrow was purchased commercially (AllCells, LLC, Berkeley, CA). Patient samples were collected from patients with newly diagnosed chronic phase CML before treatment with imatinib. Cytoreductive therapy at the time of bone marrow harvest consisted of hydroxyurea only. Approval was obtained from the Oregon Health and Science University Institutional Review Board (Portland, OR) for these studies. Informed consent was provided according to the Declaration of Helsinki. Ficoll-Hypaque-separated cells (Amersham Pharmacia, Uppsala, Sweden) were plated at 5 x 104/mL to 1 x 105/mL density with CT32228 at graded concentrations. Short-term treatment was done in RPMI 1640 containing 10% fetal bovine serum in either the presence or absence of cytokines (IL-3, GM-CSF, SCF, and +/–EPO) with graded concentrations of CT32228. Cytokine concentrations were as given for the semisolid medium protocol (see above). After 3 days, cells were washed and subsequently plated in duplicate in methylcellulose medium as described above. After a 2-week incubation at 37°C, BFU-E and CFU-GM colonies were counted. Results were calculated as the percentage of control. Statistical significance of differences was derived using the t test (SPSS software, SPSS, Inc., Chicago, IL).
Measurement of cell proliferation. The tetrazolium-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or 3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assays were done as described previously (26). Exponentially growing cells were plated in triplicate or quadruplicate at 5 x 103 per well and exposed to escalating doses of each drug independently or in combination. Cell proliferation was measured with MTT-based (Sigma, St. Louis, MO) or MTS-based viability assay (CellTiter 96 Aqueous One Solution Reagent, Promega, Madison, WI) at 24-hour intervals under exponential growth conditions. Means and SDs generated from three to four independent experiments are reported as the percentage of growth versus control at 72 hours. The IC50 and IC80 values were derived manually from the dose-response curve generated by Microsoft Excel.
Combination studies were designed according to Chou and Talalay (27): cell lines were incubated with increasing doses of drug dilutions or combinations of drug dilutions. Calcusyn software (Biosoft, Cambridge, United Kingdom) was used to calculate the combination index at different levels of growth inhibition as a quantitative measure of the degree of drug interaction. Combination index values greater than 1 indicate antagonism, values equal to 1 indicate additivity, and values lower than 1 indicate synergy. Combined drugs were used at fixed molar ratios to accommodate software requirements. Equitoxic drug doses that produced 50% of growth inhibition in single-agent experiments were chosen to determine an appropriate fixed molar ratio of 2 combined drugs.
Immunoblotting. K562 cells were cultured with increasing concentrations of CT32228 or with 0.01% DMSO as solvent control for up to 72 hours, and aliquots of cultures were harvested at certain intervals. For immunoblot analysis of Bcr-Abl, ERK and phosphorylated ERK, total phosphotyrosine, and Akt and phosphorylated Akt, the cells were washed twice with cold PBS and lysed in 1% NP40, 150 mmol/L NaCl, 20 mmol/L Tris [tris(hydroxylmethyl)aminomethane (pH 8.0)], 10% glycerol, and 1 mmol/L EDTA containing 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L orthovanadate, and 10 µg/mL aprotinin. The Abl antibody (clone 24-11) was from Santa Cruz Biotechnology (Santa Cruz, CA); the phosphotyrosine (4G10) was from Upstate Biotechnology (Lake Placid, NY); ERK, phosphorylated ERK, Akt, and phosphorylated Akt antibodies were from Cell Signaling Technology (Beverly, MA); and caspase-3, caspase-8, and caspase-9 antibodies were from BD Biosciences (Palo Alto, CA). The antibody detecting poly(ADP-ribose) polymerase (PARP) cleavage products was purchased from Upstate Biotechnology. Immunoblots were probed with a secondary horseradish peroxidase–conjugated antibody (Promega) and developed with the enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, IL). Autoradiographs were scanned with a LumiImager (Boehringer Mannheim, Mannheim, Germany).
Cell cycle analysis. K562 cells were incubated in the presence of CT32228 or DMSO only for up to 48 hours. Cells were fixed and stained according to the manufacturer's instructions and analyzed on a Guava Technologies Personal Cell Analysis instrument equipped with Cytosoft software (Guava Technologies, Hayward, CA). The relative percentages of cells in G1, S, or G2-M phase were calculated from FL-2 histograms using ModFit LT software.
Detection of apoptosis and activated caspase-3. K562 cells were cultured at 105/mL and incubated in the presence of different concentrations of CT32228 or with DMSO alone, as a control, for 72 hours. The cells were harvested, stained with Alexa Fluor 488 Annexin V and propidium iodide (PI; Molecular Probes, Eugene, OR) according to the manufacturer's instructions, and analyzed on a Becton Dickinson (San Jose, CA) FACSAria flow cytometer. Results based on two independent triplicate experiments are reported as averages ± SE. For flow cytometric detection of activated caspase-3, K562 cells were cultured at 105/mL and incubated in the presence of different concentrations of CT32228 or with DMSO alone, as a control. The cells were harvested and analyzed at 24, 48, and 72 hours according to the manufacturer's instructions (Caspase-3 Detection kit, Calbiochem, San Diego CA). Results based on three independent experiments are reported as averages ± SE.
Additionally, cytospin slides of the respective samples were analyzed by Giemsa staining (28).
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Effects of CT32228 on primary hematopoietic cells. Next, we investigated the effects of CT32228 on primary hematopoietic cells from CML patients and healthy bone marrow donors. In the first set of experiments, we assessed colony formation by mononuclear cells grown in semisolid culture in the continuous presence of CT32228. The compound reduced formation of CFU-GM and BFU-E colonies by normal mononuclear cells in a dose-dependent manner, with IC50 and IC80 values 2 to 10 times higher than for cell lines. However, there was no significant difference between CML patients and healthy donors (Fig. 2A
; Table 1B). Because previous studies had shown that inhibition of LPAAT-ß activity may be more toxic to proliferating cells than to resting cells (9), we sought to exploit the known capacity of CML progenitor cells to enter the cell cycle in the absence of cytokines (29). Mononuclear cells from CML patients and healthy individuals were cultured in graded concentrations of CT32228, with or without added cytokines. After 72 hours, the cells were plated in semisolid medium supplemented with cytokines, and CFU-GM and BFU-E were counted after 14 days. At 0.125 µmol/L CT32228, the lowest concentration used in these experiments, BFU-E and CFU-GM derived from CML patients were reduced to 18% and 10% of controls, respectively, whereas colony formation by normal MNC was much less affected (60% of controls; P = 0.0001). Moreover, whereas there was practically complete inhibition of colony formation by CML cells at concentrations above 0.5 µmol/L, inhibition of normal colonies plateaued at 70% at concentrations of 0.5 µmol/L, with no increase in toxicity at higher concentrations (Fig. 2B; Table 1B).
Effects of CT32228 on cell lines resistant to imatinib. Resistance to imatinib as a result of increased Bcr-Abl expression or mutations in the kinase domain is a significant clinical problem (30). We therefore tested the activity of CT32228 against cell lines representing these common mechanisms of imatinib resistance. Our panel included BaF3 cells expressing several Bcr-Abl kinase domain mutants frequently detected in patients (BaF/BCR-ABLY253F, BaF/BCR-ABLT315I, BaF/BCR-ABLM351T, and BaF/BCR-ABLH396R)(31) and sublines with significantly increased levels of Bcr-Abl (AR230-r1 and BaF3/p210-r1)(25). Only AR230-r1 cells were less sensitive to CT32228 than the parental line, whereas all others showed very similar sensitivity (Table 1).
LPAAT-ß inhibitors may eventually be combined with imatinib to overcome drug resistance. We therefore tested the combination of imatinib and CT32228 for its activity in BaF/3 cells expressing wild-type Bcr-Abl, the T315I and M351T mutants, as well as a subclone with increased protein expression. We observed additive to synergistic effects in all cell lines with residual sensitivity to imatinib, but not in the T315I mutant, which is completely resistant to the drug (Fig. 3 ; Table 2 ). These results suggest that combinations of LPAAT-ß inhibitors and imatinib may be clinically beneficial in patients with imatinib resistance, as long as there is residual sensitivity to imatinib. This is consistent with our previous observations (26).
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50% of the cells were in G2-M, and an increase of cells in the sub-G1 (hypodiploid) compartment was evident, indicating cell death (Fig. 4). Giemsa staining of K562 cells treated with 115 nmol/L CT32228 for 24 hours showed morphologic evidence of mitotic catastrophe as well as apoptosis (Fig. 5A
). To verify specificity of the cell cycle effect, this experiment was also done using another CML cell line (Lama84). As seen with K562 cells, treatment with CT32228 leads to G2-M arrest of Lama84 cells with similar kinetics shown with K562 (data not shown).
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12%, whereas caspase-3 activation became detectable after 48 hours. Immunoblot detection of cleaved PARP, a signaling marker for caspase-dependent apoptosis and, as recently recognized (32), caspase-independent apoptosis, was positive after 24 hours (Fig. 5C). CT32228 inhibits MAPK signaling in BCR-ABL-positive cells. To exclude any nonspecific effects of CT32228 on Bcr-Abl kinase activity or protein levels, we did immunoblots on lysates from K562 cells treated with graded concentrations of CT32228 for up to 48 hours. As expected, CT32228 did not affect Bcr-Abl protein levels or phosphorylation (Fig. 6 ). We next examined effects of CT32228 on pathways downstream of Bcr-Abl. CT32228 has been shown to inhibit translocation of Raf1 to the plasma membrane (9). We reasoned that this might interfere with signal transduction from Ras to MAPK. Consistent with this hypothesis, a significant reduction of ERK phosphorylation was detectable at 24 hours and was more pronounced with higher concentrations of CT32228 (Fig. 6). At 48 hours, phosphorylation was partially restored in cells treated with 56 nmol/L CT32228, the concentration corresponding to the IC50 in cell proliferation assays. In contrast to ERK, phosphorylation of Akt was not altered by treatment with CT32228, suggesting that this compound does not affect signaling via the phosphatidylinositol 3-kinase pathway in BCR-ABL-positive cells (Fig. 6).
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Discussion |
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Effective and tolerable treatment of leukemia depends on differential activity of the antileukemic agent, which should preferentially kill leukemic cells while sparing normal tissues from cytotoxic effects. Our experiments using short-term liquid cultures followed by colony-forming assays reveal differential activity of CT32228 in CML cells compared with normal progenitor cells only when cultured in the absence of growth factors. Under these conditions, normal cells were protected, whereas in the presence of cytokines, no differential effect was observed. Of note, at increasing concentrations of CT32228, growth factor-independent CML progenitor colonies were practically eradicated, whereas healthy progenitor colonies plateaued at 70% growth inhibition. The difference seen in the absence of cytokines may reflect the fact that only CML cells are capable of entering the cell cycle in the absence of cytokines and could evidently be exploited for in vitro purging of CML bone marrow (29). Of note, Hideshima et al. (18) reported that a CT32228 analogue had only minor effects on the growth of normal mononuclear cells. Whether a differential effect of CT32228 against CML versus normal progenitor cells will be seen in vivo may ultimately depend on local cytokine concentrations in the bone marrow.
Combinations of CT32228 with imatinib revealed additivity or synergism both in imatinib-sensitive and imatinib-resistant cell lines. However, this was limited to cells with residual sensitivity (i.e., sensitive to dose escalation) to imatinib, whereas no synergism or additivity was seen in the T315I mutant (i.e., insensitive to dose escalation). These data are consistent with our previous observations using combinations of imatinib and decitabine or arsenic trioxide (26). Taken together, the results of the present study show that CT32228 has considerable antileukemic activity as a single agent and in combination with imatinib.
Inhibition of proliferation of K562 cells by CT32228 was associated with blockade of the cell cycle in G2-M (Fig. 4). This observation is in line with data reported by Hideshima et al. (18), who analyzed CT32228-related compounds with anti-LPAAT-ß activity in multiple myeloma cells. As early as 24 hours after start of treatment with CT32228, K562 showed morphologic changes consistent with mitotic catastrophe, a finding that is consistent with the cell cycle analysis. The mechanism responsible for the G2-M arrest in the K562 cells is unclear. Hideshima et al. (18) suggested that the G2-M arrest may be triggered through a p53-dependent DNA damage response involving up-regulation of p21. However, as K562 cells lack functional p53 (33), this mechanism cannot account for the G2-M arrest in this cell line. Alternatively, a p53-independent mechanism via p14(ARF) may account for the G2-M arrest in our hands (34). Further experiments will be necessary to determine the underlying mechanism.
FACS analysis with Annexin V/PI-negative staining showed that K562 cells treated with CT32228 die via apoptosis (Fig. 5). Consistent with this, we were able to show cleavage/activation of caspase-3 and cleavage of PARP. It should be noted that the extent of caspase-3 cleavage, detectable by FACS but not on immunoblots, was disproportional and delayed in relation to the significant percentage of apoptotic cells appearing as early as 24 hours after start of treatment. This suggests that a caspase-3-independent mechanism must be responsible for apoptosis induction, at least early after initiating treatment. In line with this observation, cleavage of PARP, a readout molecule for caspase-dependent and caspase-independent apoptosis, was detectable before caspase-3 activation (32).
Because lysophosphatidic acid is required for the activation of Raf1, a key intermediate in the Ras/MAPK pathway, depletion of lysophosphatidic acid by LPAAT-ß inhibition was expected to inhibit ERK1/2. Consistent with this, ERK1/2 phosphorylation was reduced in cells exposed to CT32228 (Fig. 6). As activation of MAPK signaling has been reported in response to imatinib treatment as a potential resistance-inducing pathway (16), inhibitors that counteract MAPK activity may be of clinical value to overcome imatinib resistance. In contrast to ERK inhibition, LPAAT-ß inhibition did not affect phosphatidylinositol 3-kinase/Akt signaling in K562 cells (Fig. 6), although Coon et al. (17) observed a significant effect on Akt signaling in vascular smooth muscle cells and a minor effect in a lymphoma cell line. The biochemical basis for this apparent cell line specificity remains elusive.
In summary our data implicate LPAAT-ß as a therapeutic target in BCR-ABL-positive leukemias. Although the clinical usefulness of CT32228 is limited by its low bioavailability due to hydrophobicity, the development of compounds with more favorable pharmacokinetic properties is ongoing. Given the excellent activity of CT32228 in solid tumor cell lines (9), inhibition of LPAAT-ß may be a broadly applicable therapeutic strategy in malignant diseases.
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: P. La Rosée and T. Jia contributed equally to this article.
Received 1/23/06; revised 7/30/06; accepted 8/24/06.
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; untreated or on hydroxyurea, n = 5) or from healthy donors (
; n = 5) were assayed in semisolid medium in the continuous presence of increasing concentrations of CT32228 (A) or after short-term drug exposure in liquid medium without growth factors and subsequent culture in growth factor containing semisolid medium without drug (B). Depending on growth factors added, CFU-GM (left) and BFU-E (right) were counted. Statistical analysis using the t test revealed a significant differential growth-inhibitory activity of CT32228 against CML progenitors that grow in the absence of growth factors (B). Data points represent mean of duplicate analysis and are given as percentage growth inhibition versus untreated control (Y axis).
p-Tyr), Abl and BCR-ABL (