- Email: [email protected]
Constitutive phosphorylation of the S6 ribosomal protein via mTOR and ERK signaling in the peripheral blasts of acute leukemia patients
Experimental Hematology 34 (2006) 1182–1190
Constitutive phosphorylation of the S6 ribosomal protein via mTOR and ERK signaling in the peripheral blasts of acute leukemia patients Sue Chowa, Mark D. Mindenb,c, and David W. Hedleya,c a
Division of Applied Molecular Oncology, Ontario Cancer Institute, University of Toronto, Toronto, Canada; Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, University of Toronto, Toronto, Canada; c Department of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, Canada
b
(Received 27 January 2006; revised 27 April 2006; accepted 1 May 2006)
Objective. The phosphorylation state of the S6 ribosomal protein was measured in the peripheral blasts of 19 newly diagnosed patients with acute leukemia. Methods. We employed a flow cytometry protocol that enabled correlated measurement of pS6, phosphorylation of extracellular signal-regulated kinase (pERK), and cluster differentiation surface markers. Baseline levels of pS6 in leukemic blasts were compared with those found when the samples were activated using stem cell factor, or exposed to rapamycin, LY294002, or the mitogen-activated protein kinase inhibitor U0126. Results. Results showed a considerable degree of intra- and intertumoral heterogeneity in the constitutive levels of pS6. Rapamycin and LY294002 suppressed pS6 in 10 of 11 cases that showed increased basal levels, consistent with phosphatidylinositol 3 (PI3)-kinase/Akt/ mTOR signaling being the predominant upstream signaling pathway. However, in 6 of 11 cases pS6 was also suppressed by U0126, indicating that the ERK pathway can significantly input to pS6. Conclusions. The constitutive activation of pS6 in acute leukemia patients likely reflects alterations in growth factor signaling that can be mediated by the ERK as well as the mTOR pathway, and could potentially have prognostic significance. As well as identifying aberrant signal transduction in leukemia patients, the flow cytometry methodology has potential for the pharmacodynamic monitoring of novel agents that inhibit ERK or PI3-kinase/Akt/ mTOR signaling. Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc.
The importance of aberrant signal transduction in the development and progression of cancers including acute leukemia is well recognized, although the detailed regulation of signaling networks in relation to oncogenic mutations remains incompletely understood. HoweveroHOwH, this subject is becoming relevant to clinical oncology due to the rapid development of pharmacological agents that target signaling pathways [1–5]. With the notable exception of chronic myelogenous leukemia, where bcr/abl kinase inhibitors are highly specific and effective [6], the clinical application of signal transduction inhibitors is challenging due
Offprint requests to: David W. Hedley, M.D., Department of Medical Oncology and Hematology, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada; E-mail: david.hedley@ uhn.on.ca
to the complexity of signaling pathways, the presence of multiple genetic abnormalities in most cases, and the problem of tumoral heterogeneity. To address these problems, analytical methods are needed that can be applied to clinical samples in order to identify if the drug target is being expressed in that patient, as well as allowing pharmacodynamic monitoring to determine if drug treatment results in the inhibition of its downstream targets. In concert with ongoing research in basic science and drug design, this approach is expected to facilitate the rational development of molecular cancer therapeutics, including individualized patient treatment. Recently, flow cytometry has emerged as a powerful technique for the study of signaling pathways, based on the combined use of phenotypic markers to identify cell type and phosphospecific antibodies to determine the activation states of signal transduction elements [7–10]. This
0301-472X/06 $–see front matter. Copyright Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2006.05.002
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
technique is particularly well suited to the study of leukemia, and there is a growing number of reports showing that aberrant signaling mechanisms can be identified in patient samples by flow cytometry [8,11,12]. Our own interest in this subject has focused on the development and clinical application of techniques for the pharmacodynamic monitoring of signal transduction inhibitors in early phase clinical trials, with an emphasis on agents that target the extracellular-regulated kinase (ERK) pathway [7,13]. The ERK pathway interacts with a second major signaling pathway centered on the mammalian target of rapamycin (mTOR) to regulate important elements involved in protein translation [14,15]. The mTOR pathway is highly conserved, and in primitive organisms appears to be exclusively involved in the regulation of translation. It is sensitive to nutrient and oxygen levels, as well as to upstream signaling pathways, including phosphatidylinositol-3-kinase/Akt [16–19]. The activity of mTOR facilitates protein translation by two distinct mechanisms. The first involves the phosphorylation of an inhibitory protein, 4E-BP1, allowing release of the eukaryotic initiation factor-4E that can then assemble in the translation initiation complex [14,20]. The second downstream target of mTOR is p70S6kinase, whose substrate is the S6 ribosomal protein; a component of the S40 ribosome subunit [21]. Interestingly, both of these pathways downstream from mTOR are positively regulated by ERK, while there is recent evidence that ERK can also regulate upstream of mTOR through phosphorylation of TSC2 [21,22]. This suggests extensive potential for fine tuning, including the coordination of cell growth and cell-cycle regulation. In higher organisms, the mTOR pathway plays additional roles, including cell-cycle regulation and maintenance of cell survival, and it is therefore an attractive target for novel anticancer agents [19,23,24]. This is supported by preclinical data using rapamycin and its analogues, which suggest a broad range of anticancer activity, and also by early clinical trial data, including evidence for activity in acute myeloid leukemia (AML) patients [25]. It is, therefore, relevant to ask if mTOR and its targets are constitutively activated in cancer patients, and also to determine the extent to which this is further enhanced by inputs from the ERK pathway.
Materials and methods Patients These were newly diagnosed acute leukemia patients being treated at the Princess Margaret Hospital, Toronto. Following preliminary work to optimize the flow cytometry technique, a total of 19 patients were included in the present series. These were selected based on the presence of circulating blast cells. According to institutional research ethics guidelines, 5 mL peripheral blood was obtained, using heparin as the anticoagulant, and processed within 24 hours in the flow cytometry laboratory. In addition to the leukemia patient samples, we also examined granulocyte colony-
1183
stimulating factor (G-CSF) mobilized CD34þ blasts in peripheral blood samples obtained from allogeneic stem cell transplant donors. Reagents and antibodies Wash buffer consisted of calcium- and magnesium-free phosphate-buffered saline containing 4% fetal bovine serum. Phorbol myristate acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) was diluted to a 40 mM working solution from a 40 mM stock in ethanol. Stem cell factor (SCF) was obtained from Calbiochem (San Diego, CA, USA), and diluted to 10 mg/mL in wash buffer. Insulin-like growth factor-1 (IGF-1) was obtained from PeproTech (Rocky Hill, NJ, USA) and made up to 10 mg/mL in wash buffer. The mitogen-activated protein kinase kinase (MEK) inhibitor U0126 (Cell Signaling Technology, Beverly, MA, USA) was prepared as a 10-mM solution in methanol. Rapamycin (Calbiochem) was diluted to a 100 mg/mL working solution from a 1 mg/mL stock in dimethylsulfoxide. LY294002 (Calbiochem) was prepared as a 50-mM solution in ethanol. A rabbit polyclonal antibody to pS6 Ser 235/236 (#2211), rabbit monoclonal antibody to pAkt Ser 473 (#4058), and a mouse monoclonal anti-phosphorylation of extracellular signal-regulated kinase (pERK) 1/2 (Thr 202/Tyr 204; #9106) conjugated to Alexa Fluor 488 were obtained from Cell Signaling Technology. Surface markers CD34-phycoerythrin (PE) (clone 581) and CD45-PE (clone J.33) were obtained from Beckman Coulter, Inc. (Miami, FL, USA). Alexa Fluor 647-conjugated goat anti-rabbit-IgG F(ab0 )2 was purchased from Molecular Probes (Eugene, OR, USA). Flow cytometry protocol Figure 1 gives a schematic of the interactions between mTOR and ERK that are relevant to this work. The flow cytometry protocol analyzed these interactions by measuring the basal levels of pS6 and pERK in the leukemic blast population, and how these alter in response to agents that inhibit upstream signaling elements, as well as to ex vivo activation via c-Kit using SCF. Whole blood samples were divided into 200 mL aliquots in 5 mL round-bottomed polypropylene tubes, and placed in a 37 C dry bath. To one set of tubes, inhibitors (100 mM U0126, 1 mg/mL Rapamycin, or 500 mM LY294002) were added and incubated for 30 minutes. A second set of tubes was pretreated with inhibitors for 25 minutes, and then activated by adding SCF 100 ng/mL for 5 minutes. As a positive control for ERK activation, 400 nM PMA was added to an additional sample for 10 minutes. Previous work from our laboratory ([7] and unpublished data) has found that PMA strongly activates ERK in all nucleated blood cells, regardless of whether these are able to respond to SCF stimulation. Following treatment, samples were processed using a protocol for fixation, red cell lysis, and permeabilization that was recently developed in our laboratory, and optimizes the preservation of phenotypic features as well as intracellular phosphorylated epitopes in whole blood samples [26]. Briefly, samples were removed from the dry bath, and fixed by adding methanol-free formaldehyde (10% solution; Polysciences, Warrington, PA, USA) to give a final concentration of 4% for 10 minutes. Then 2 mL 0.1165% Triton X-100 (final concentration 0.1%) was added to lyse red blood cells. After additional incubation for 30 minutes at room temperature, 2 mL cold wash buffer was added. Cells were centrifuged for 3 minutes at 1000g at 10 –15 C. The
1184
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
Figure 1. Schematic showing major pathways and sites of action of agents used in this study. eIF-4E 5 eukaryotic initiation factor-4E; ERK 5 extracellular signal-regulated kinase; MEK 5 mitogen-activated protein kinase kinase; mTOR 5 mammalian target of rapamycin; PI3 5 phosphatidylinositol 3; SCF 5 stem cell factor.
supernatant was aspirated, and the cell pellet resuspended in 1 mL cold freezing medium consisting of 10% glycerol, 20% fetal bovine serum in RPMI 1640 tissue culture medium, and stored at 20 C until antibody staining.
525, 575, and 675 nM. Fluorescence compensation was not used. Listmode analysis was done with FlowJo software (Tree Star, Inc., Ashland, OR, USA).
Antibody staining As soon as frozen samples were thawed, aliquots containing 0.5 million cells were diluted into 2 mL cold wash buffer then centrifuged. Cells were resuspended in 1 mL 50% ice-cold methanol in 0.9% NaCl, and held on ice for 10 minutes. After removal of methanol, cells were washed with 2 mL wash buffer, and the cell pellet resuspended and labeled with either 0.5 mL unconjugated anti-pS6 Ser 235/236 or 1 mL unconjugated anti-Akt Ser 473 for 15 minutes at room temperature. Samples were washed and then stained with an antibody cocktail consisting of 10 mL Alexa Fluor 488-conjugated anti-pERK, the Alexa Fluor 647conjugated secondary antibody, and PE-conjugated anti-CD34. In acute leukemia cases, that were CD34, the combination of CD45-PE staining intensity and orthogonal light scatter was substituted in order to identify leukemic blasts [27]. After 15 minutes incubation at room temperature, samples were washed and resuspended in 150 mL wash buffer for flow cytometry.
Results and discussion
Flow cytometry This was done using a modified Epics Elite cell sorter (Beckman Coulter, Inc., Miami, FL, USA), with spatially separated aircooled argon and HeNe lasers, each emitting at 20 mW. Fluorescence signals were collected through bandpass filters centered at
Activation of S6 in mobilized peripheral stem cells from normal donors Mobilized peripheral stem cells were included in the study in order to gain insight into S6 activation pathways in genotypically normal CD34þ blasts in peripheral blood samples. A total of four cases were examined; the results were very similar in all of these. As shown in Figure 2, the sample preparation protocol gave excellent preservation of CD34 staining and orthogonal light scatter, allowing unambiguous identification of the blast cell population. Bivariate plots of pERK vs pS6 showed that fluorescence intensities for the blast cells were within a narrow range of values. There was no discernable decrease in either pERK or pS6 following preincubation with rapamycin, LY294002, or U0126 (data not shown), suggesting that the fluorescence signals represent background due to autofluorescence and nonspecific antibody binding. Treatment with SCF resulted in an increase in both pERK and pS6 in the blast population, as illustrated in Figure 2. In
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
1185
Figure 2. Monitoring signaling pathways in granulocyte colony-stimulating factor–mobilized peripheral stem cells. The sample preparation technique readily identifies these, based on light scatter and CD34 surface staining (top panels). Lower left panels show bivariate dot plots of pS6 vs phosphorylation of extracellular signal-regulated kinase (ERK), and responses to the indicated treatments. Note that both stem cell factor (SCF) and phorbol myristate acetate (PMA) activate ERK and S6, whereas insulin-like growth factor-1 (IGF-1) has no significant effect. The effects of SCF and PMA (not shown) on ERK and S6 are inhibited by U0126 but not by rapamycin, indicating that acute activation of S6 is predominantly mediated by the ERK pathway. Bottom right panels show Ser473 Akt phosphorylation on the vertical axis. Note that whereas SCF activates both ERK and Akt, PMA activates ERK but not Akt, and IGF-1 activates Akt but not ERK. Quadrants define labeling intensities of the unstimulated control samples, and are placed to aid visual inspection.
contrast, stimulation using IGF-1 resulted in activation of Akt with minimal effects of ERK, and did not phosphorylate S6 (Fig. 2). Treatment with PMA activated pERK and pS6 but not pAkt in CD34þ blasts, as well as in the lymphocyte, neutrophil, and monocyte populations. Pretreatment with U0126 inhibited activation of both pERK and pS6 by SCF and PMA, whereas rapamycin was ineffective. Taken together, these results indicate low or absent basal levels of ERK and S6 phosphorylation in mobilized peripheral stem cells. When these cells are acutely activated by SCF, ERK rather than mTOR appears to be the predominant pathway for serine 235/236 phosphorylation of S6.
Constitutive S6 phosphorylation in the peripheral blasts of acute leukemia patients Table 1 gives a summary of the patient demographics, including the type of acute leukemia, cytogenetic analysis, and clinical outcome. Six patients received either no chemotherapy or low-dose palliative treatment because of age or other medical conditions. Of those treated with intensive chemotherapy, 10 achieved hematological complete remission, and the remainder showed gross evidence of residual disease in the bone marrow after chemotherapy. Patients were selected for study based on the presence of at least 5% circulating blast cells. However, because of the excellent preservation of light scatter and surface
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
1186 Table 1. Patient information
Patient no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Type
Cytogenetics
Age (y)
Increased basal pS6
Rapamycinsensitive
LY294002sensitive
U0126sensitive
SCFresponse
Clinical outcome
AML (M2) ALL (Ph-ve) MDS/AML AML (M2) AML (M2) MDS/AML AML (M2) ALL (Ph-ve) AML (M4) AML (M7) AML (M0) MDS/AML AML (M2) AML (M2) PRV/AML AML (M4) ALL (Phþve) APL (M3) AML (M2)
46XY, inv(20) (q12q31.1)
38 42 72 44 72 67 25 47 26 46 80 76 77 79 62 48 74 45 18
þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ
þ þ þ þ þ þ
þ þ þ þ
þ
þ
þ þ þ þ
þ þ þ þ
þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
CR CR Palliative CR Palliative Palliative CR Palliative CR Progressed Palliative Palliative Progressed CR CR CR CR CR Progressed
47XY, þ13 46XX (Flt3 ITD) 46XY add (6p) 45XY, -7 46XX, t(8;21) 46XY, t(8;21) 46XY 46XY 46XX 46XX 46XY 46XY 46XX, t(9;22) 46XX, t(15;17) 4XX
ALL 5 acute lymphocytic leukemia; AML 5 acute myeloid leukemia; APL 5 acute promyelocytic leukemia; CR 5 complete response; MDS 5 myelodysplastic syndrome; PRV 5 polycythemia rubra vera; SCF 5 stem cell factor.
immunophenotype achieved by the flow cytometry protocol, we found that blast populations that were !1% of the total white blood cell count could be readily identified. Figure 3 illustrates examples of the patterns of signaling pathway activation, as well as the gating strategy based on light scatter and surface immunophenotype used to identify the blast population. In contrast to mobilized peripheral stem cells, rapamycin, LY294002, and U0126 produced significant decreases in S6 phosphorylation in the blast population, as summarized in Tables 1 and 2. Typically S6 phosphorylation showed considerable cellular heterogeneity within individual patients. For example, patient no. 6 (Fig. 3) shows a range in basal levels of pS6 of more than 100-fold. With one exception, rapamycin suppressed pS6 levels in those samples showing constitutive activation, as a result of which, the range of individual cell values became much narrower. Taking into account the often extreme cellular heterogeneity and responses to inhibitors, we categorized 11 of 19 samples as showing elevated pS6 levels (Table 1). Because rapamycin is a highly specific inhibitor of mTOR, these results suggest that the mTOR pathway is active in approximately half of acute leukemia patients. The phosphatidylinositol 3 (PI3)-kinase/Akt pathway is believed to be an important upstream activator of mTOR. Although Akt can directly phosphorylate mTOR, the significance of this is not certain, and mTOR activation appears to occur by a more circuitous route involving other signaling elements including tuberin (TSC2) and the small GTP-binding protein Rheb [18,28,29]. In preliminary experiments using a rabbit monoclonal antibody to Akt
serine 473, we found increased levels of expression in some samples showing elevated S6 phosphorylation, suggesting that constitutive Akt activation is involved in the rapamycin-sensitive phosphorylation of S6 in some acute leukemia patients (Fig. 4). This is further supported by the inhibitory effects on pS6 after treatment with the PI3-kinase inhibitor LY294002, although it should be noted that LY294002 and the structurally unrelated PI3-kinase inhibitor wortmannin both have been reported to inhibit mTOR [30]. Therefore, we cannot exclude the possibility that the effects of LY294002 on pS6 could be due to mTOR rather than to PI3-kinase inhibition. Effects of U0126 Constitutive pERK has been reported to occur in some AML patients [12,31]. However, in contrast to the extensive cellular heterogeneity seen with pS6, baseline pERK levels in the peripheral blast populations were narrowly distributed, and in all cases showed mean values that were only slightly greater than background autofluorescence. Furthermore, as summarized in Table 3, although there was a small decrease in the mean level of pERK in samples pretreated with U0126 (0.63 0.41 vs 0.51 0.40 arbitrary units), this was not statistically significant. Nevertheless, treatment with U0126 resulted in a significant decrease in the mean pS6 levels in the series of 19 samples as shown in Tables 1 and 2. By visual inspection of the pERK vs pS6 bivariate dot plots this was evident in 6 of the 11 samples that contained high pS6 cells (Table 1, Fig. 3). It should be noted that the concentration of U0126 used in these experiments was approximately 10-fold greater than that typically used
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
1187
Figure 3. Bivariate plots of pS6 vs phosphorylation of extracellular signal-regulated kinase (pERK) obtained from six acute myeloid leukemia (AML) patients, illustrating the effects of treatment with rapamycin, LY294002, and U0126 vs untreated control. Left panels show the respective plots of orthogonal light scatter vs CD34 or CD45, and illustrate the gating regions used to identify the blast population. Patient identifier is given in the lower right corner of these plots. Note extreme heterogeneity seen in pS6 levels in patient nos. 3, 6, and 14. This is strongly inhibited by rapamycin and LY294002, and to a lesser extent by U0126. PE 5 phycoerythrin.
to inhibit MEK in vitro, but this was based on our previous experience and is needed because of greater protein binding of U0126 in whole blood [7]. Because U0126 is considered to be a selective MEK inhibitor, we suggest that the effects on S6 phosphorylation in acute leukemia patient samples indicate the presence of low levels of constitutive pERK activity that, in concert with mTOR signaling, are able to enhance S6 phosphorylation. This effect is clearly seen in Figure 5 (patient no. 5), where the blast population defined by CD34 and side scatter
shows greater inhibition of pS6 with U0126 compared with rapamycin, and is even more pronounced in a CD34/high side-scatter subpopulation, where the cells with the greatest levels of pS6 also show high levels of pERK that are inhibited by U0126. Effects of SCF on AML blasts Table 3 summarizes the effects of stimulation with SCF and PMA on mean pERK levels. In 15 of 19 patients ex vivo treatment with SCF produced acute activation of signaling
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
1188
Table 2. Mean P-S6 values for blast populations
Table 3. Mean pERK values for blast populations
Untreated
Rapamycin
LY294002
U0126
15.0 11.5
7.8 4.3*
7.6 4.3**
9.7 6.1***
*p 5 0.006; **p 5 0.007; ***p 5 0.013; two-tailed paired t-test; comparing to untreated control.
pathways and, in 14 cases, this resulted in increases in both ERK and S6 phosphorylation, similar to the example shown in Figure 5. In the remaining case, there was no effect on ERK or S6, but there was a large increase in Akt phosphorylation. As shown in Table 1, all of the AML cases were responsive to SCF, whereas stimulation was not seen in any of the three cases of acute lymphocytic leukemia or the single case of acute promyelocytic leukemia. Similar to results obtained with mobilized peripheral stem cells, acute activation of S6 after SCF treatment was inhibited by U0126 but not rapamycin; this was also seen with PMA activation. In contrast to the constitutive S6 phosphorylation seen in AML blasts, where mTOR signaling predominates, acute activation via SCF occurs through the ERK pathway. Conclusions Overall, the results described here support the recent report by Irish et al. [8] that flow cytometry is able to reveal signaling complexity in AML patient samples. In this article, we used a recently developed whole-blood fixation technique that allows measurement of constitutive phosphoryla-
Untreated 0.63 0.41
U0126
SCF
PMA
0.51 0.4
2.54 3.23
7.94 3.35
pERK 5 phosphorylation of extracellular signal-regulated kinase; PMA 5 phorbol myristate acetate; SCF 5 stem cell factor.
tion states, as well as the response to ex vivo treatment with growth factors and inhibitors of signaling pathways [26]. This allowed identification of high levels of constitutive phosphorylation of the S6 ribosomal protein in more than half of AML patients tested, as well as revealing a complex interaction between the Akt/mTOR and ERK signaling pathways that appears to show considerable inter-patient heterogeneity. These effects were not seen in mobilized peripheral stem cells from normal donors, and most likely reflect alterations in upstream signaling elements, such as N-ras or flt-3 that occur as part of the leukemic process. Based on the overall greater effect of rapamycin compared with U0126, we propose that in most cases of AML, mTOR is the predominant mediator of constitutive S6 phosphorylation. However, in the example shown in Figure 5, S6 phosphorylation is driven by ERK signaling, and this case is relatively refractory to rapamycin, suggesting that AML patients might be stratified based on constitutive activation of the upstream pathways. This would have implications for selection of drugs targeting different signaling molecules. In contrast to the predominance of mTOR in constitutive
Figure 4. Dual labeling of pAkt (S473) and phosphorylation of extracellular signal-regulated kinase (pERK) in patient no. 3 (top panels), compared with pS6 vs pERK (lower panels). Similar to pS6, pAkt shows a heterogeneous distribution in the untreated control. Preincubation with LY294002 results in decreased pAkt, whereas stem cell factor (SCF) acutely activates pAkt as well as pERK in all of the CD34þ blasts.
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
1189
Figure 5. Constitutive of extracellular signal-regulated kinase (ERK) activation of S6 in a subpopulation defined by increased orthogonal light scatter and loss of CD34 expression. As seen in the lower right panel, this population is responsive to stem cell factor (SCF), indicating that it is likely of leukemic origin. Note the great heterogeneity of pS6 in these cells, with clear evidence for increased phosphorylation ERK (pERK) in cells at the upper extreme of pS6. In this example U0126 strongly inhibits pS6 as expected, whereas rapamycin has minimal effects. This pattern is also seen in the CD34þ population, illustrated in the upper set of panels. PE 5 phycoerythrin.
S6 phosphorylation, ERK predominates when S6 is activated by ex vivo treatment with SCF; similar to the effects seen with the mobilized normal peripheral stem cells. Given that the Akt/mTOR pathway appears to play a significant role in cell survival signaling, it will be of interest to determine if high constitutive levels are predictive of acute leukemia patient outcome, although this was not evident in the present small series, as shown in Table 1. Our original motivation for developing whole-blood flow cytometry methods to study signaling pathways was the need for pharmacodynamic monitoring of novel agents, where isolation techniques are likely to perturb the drug/target equilibrium [7]. The method described in the present ar-
ticle appears to be well-suited to meet this need, and has the potential to interrogate several signaling elements of potential importance in AML, such as c-kit, flt-3, PI3-kinase, as well as mTOR. It should also be noted that relative to previously described techniques from our own laboratory and from others, standard phenotypic features of light scatter and surface immunophenotype are well preserved in clinical samples, offering the potential to extend applications to rare events, including study of minimal residual disease. Acknowledgments The authors thank Dr. James Jacobberger, Case Western Reserve University; Dr. T. Vincent Shankey, Advanced Technology Center,
1190
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
Beckman-Coulter; and Dr. Bradley Smith, Cell Signaling Technology, for their critical comments. This work was supported by a grant from the Ontario Cancer Research Network and research collaboration support was provided by Advanced Technology Center, Beckman Coulter, Inc., Miami FL, USA.
References 1. Bernasconi P, Boni M, Cavigliano PM, et al. Molecularly targeted therapy in acute myeloid leukemia. Ann N Y Acad Sci. 2004;1028: 409–422. 2. Levis M. Recent advances in the development of small-molecule inhibitors for the treatment of acute myeloid leukemia. Curr Opin Hematol. 2005;12:55–61. 3. Sattler M, Scheijen B, Weisberg E, Griffin JD. Mutated tyrosine kinases as therapeutic targets in myeloid leukemias. Adv Exp Med Biol. 2003;532:121–140. 4. Sawyers C. Targeted cancer therapy. Nature. 2004;432(7015):294– 297. 5. Stirewalt DL, Meshinchi S, Radich JP. Molecular targets in acute myelogenous leukemia. Blood Rev. 2003;17:15–23. 6. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001;344:1031–1037. 7. Chow S, Patel H, Hedley DW. Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry. 2001;46(2):72–78. 8. Irish JM, Hovland R, Krutzik PO, et al. Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell. 2004;118:217–228. 9. Jacobberger JW, Sramkoski RM, Frisa PS, et al. Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity. Cytometry. 2003;54A(2):75–88. 10. Perez OD, Nolan GP. Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat Biotechnol. 2002;20:155–162. 11. Pallis M, Seedhouse C, Grundy M, Russell N. Flow cytometric measurement of phosphorylated STAT5 in AML: lack of specific association with FLT3 internal tandem duplications. Leuk Res. 2003;27:803–805. 12. Ricciardi MR, McQueen T, Chism D, et al. Quantitative single cell determination of ERK phosphorylation and regulation in relapsed and refractory primary acute myeloid leukemia. Leukemia. 2005;19: 1543–1549. 13. Tong FK, Chow S, Hedley DW. Pharmacodynamic monitoring of BAY 43-9006 (Sorafenib) in phase i clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells. Cytometry B Clin Cytom 2006;70:107–114. 14. Herbert TP, Tee AR, Proud CG. The extracellular signal-regulated kinase pathway regulates the phosphorylation of 4E-BP1 at multiple sites. J Biol Chem. 2002;277:11591–11596. 15. Wang L, Proud CG. Ras/Erk signaling is essential for activation of protein synthesis by Gq protein-coupled receptor agonists in adult cardiomyocytes. Circ Res. 2002;91:821–829.
16. Abraham RT. mTOR as a positive regulator of tumor cell responses to hypoxia. Curr Top Microbiol Immunol. 2004;279:299–319. 17. Alvarez B, Garrido E, Garcia-Sanz JA, Carrera AC. Phosphoinositide 3-kinase activation regulates cell division time by coordinated control of cell mass and cell cycle progression rate. J Biol Chem. 2003;278: 26466–26473. 18. Brugarolas J, Lei K, Hurley RL, Manning BD, et al. Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 2004;18:2893–2904. 19. Wendel HG, De Stanchina E, Fridman JS, et al. Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature. 2004; 428(6980):332–337. 20. Gingras AC, Gygi SP, Raught B, et al. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev. 1999;13: 1422–1437. 21. Fingar DC, Richardson CJ, Tee AR, Cheatham L, Tsou C, Blenis J. mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E. Mol Cell Biol. 2004;24:200–216. 22. Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell. 2005;121:179–193. 23. Mita MM, Mita A, Rowinsky EK. The molecular target of rapamycin (mTOR) as a therapeutic target against cancer. Cancer Biol Ther. 2003;2(suppl 1):S169–S177. 24. Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute myeloid leukemia cells requires PI3 kinase activation. Blood. 2003; 102:972–980. 25. Recher C, Beyne-Rauzy O, Demur C, et al. Antileukemic activity of rapamycin in acute myeloid leukemia. Blood. 2005;105:2527–2534. 26. Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV. Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A. 2005;67:4–17. 27. Borowitz MJ, Guenther KL, Shults KE, Stelzer GT. Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis. Am J Clin Pathol. 1993;100:534–540. 28. Garami A, Zwartkruis FJ, Nobukuni T, et al. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol Cell. 2003;11:1457–1466. 29. Nave BT, Ouwens M, Withers DJ, Alessi DR, Shepherd PR. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J. 1999; 344(pt 2):427–431. 30. Brunn GJ, Williams J, Sabers C, Wiederrecht G, Lawrence JC Jr, Abraham RT. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. 1996;15:5256–5267. 31. Kim SC, Hahn JS, Min YH, Yoo NC, Ko YW, Lee WJ. Constitutive activation of extracellular signal-regulated kinase in human acute leukemias: combined role of activation of MEK, hyperexpression of extracellular signal-regulated kinase, and downregulation of a phosphatase, PAC1. Blood. 1999;93:3893–3899.
Constitutive phosphorylation of the S6 ribosomal protein via mTOR and ERK signaling in the peripheral blasts of acute leukemia patients Sue Chowa, Mark D. Mindenb,c, and David W. Hedleya,c a
Division of Applied Molecular Oncology, Ontario Cancer Institute, University of Toronto, Toronto, Canada; Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, University of Toronto, Toronto, Canada; c Department of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, Canada
b
(Received 27 January 2006; revised 27 April 2006; accepted 1 May 2006)
Objective. The phosphorylation state of the S6 ribosomal protein was measured in the peripheral blasts of 19 newly diagnosed patients with acute leukemia. Methods. We employed a flow cytometry protocol that enabled correlated measurement of pS6, phosphorylation of extracellular signal-regulated kinase (pERK), and cluster differentiation surface markers. Baseline levels of pS6 in leukemic blasts were compared with those found when the samples were activated using stem cell factor, or exposed to rapamycin, LY294002, or the mitogen-activated protein kinase inhibitor U0126. Results. Results showed a considerable degree of intra- and intertumoral heterogeneity in the constitutive levels of pS6. Rapamycin and LY294002 suppressed pS6 in 10 of 11 cases that showed increased basal levels, consistent with phosphatidylinositol 3 (PI3)-kinase/Akt/ mTOR signaling being the predominant upstream signaling pathway. However, in 6 of 11 cases pS6 was also suppressed by U0126, indicating that the ERK pathway can significantly input to pS6. Conclusions. The constitutive activation of pS6 in acute leukemia patients likely reflects alterations in growth factor signaling that can be mediated by the ERK as well as the mTOR pathway, and could potentially have prognostic significance. As well as identifying aberrant signal transduction in leukemia patients, the flow cytometry methodology has potential for the pharmacodynamic monitoring of novel agents that inhibit ERK or PI3-kinase/Akt/ mTOR signaling. Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc.
The importance of aberrant signal transduction in the development and progression of cancers including acute leukemia is well recognized, although the detailed regulation of signaling networks in relation to oncogenic mutations remains incompletely understood. HoweveroHOwH, this subject is becoming relevant to clinical oncology due to the rapid development of pharmacological agents that target signaling pathways [1–5]. With the notable exception of chronic myelogenous leukemia, where bcr/abl kinase inhibitors are highly specific and effective [6], the clinical application of signal transduction inhibitors is challenging due
Offprint requests to: David W. Hedley, M.D., Department of Medical Oncology and Hematology, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada; E-mail: david.hedley@ uhn.on.ca
to the complexity of signaling pathways, the presence of multiple genetic abnormalities in most cases, and the problem of tumoral heterogeneity. To address these problems, analytical methods are needed that can be applied to clinical samples in order to identify if the drug target is being expressed in that patient, as well as allowing pharmacodynamic monitoring to determine if drug treatment results in the inhibition of its downstream targets. In concert with ongoing research in basic science and drug design, this approach is expected to facilitate the rational development of molecular cancer therapeutics, including individualized patient treatment. Recently, flow cytometry has emerged as a powerful technique for the study of signaling pathways, based on the combined use of phenotypic markers to identify cell type and phosphospecific antibodies to determine the activation states of signal transduction elements [7–10]. This
0301-472X/06 $–see front matter. Copyright Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2006.05.002
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
technique is particularly well suited to the study of leukemia, and there is a growing number of reports showing that aberrant signaling mechanisms can be identified in patient samples by flow cytometry [8,11,12]. Our own interest in this subject has focused on the development and clinical application of techniques for the pharmacodynamic monitoring of signal transduction inhibitors in early phase clinical trials, with an emphasis on agents that target the extracellular-regulated kinase (ERK) pathway [7,13]. The ERK pathway interacts with a second major signaling pathway centered on the mammalian target of rapamycin (mTOR) to regulate important elements involved in protein translation [14,15]. The mTOR pathway is highly conserved, and in primitive organisms appears to be exclusively involved in the regulation of translation. It is sensitive to nutrient and oxygen levels, as well as to upstream signaling pathways, including phosphatidylinositol-3-kinase/Akt [16–19]. The activity of mTOR facilitates protein translation by two distinct mechanisms. The first involves the phosphorylation of an inhibitory protein, 4E-BP1, allowing release of the eukaryotic initiation factor-4E that can then assemble in the translation initiation complex [14,20]. The second downstream target of mTOR is p70S6kinase, whose substrate is the S6 ribosomal protein; a component of the S40 ribosome subunit [21]. Interestingly, both of these pathways downstream from mTOR are positively regulated by ERK, while there is recent evidence that ERK can also regulate upstream of mTOR through phosphorylation of TSC2 [21,22]. This suggests extensive potential for fine tuning, including the coordination of cell growth and cell-cycle regulation. In higher organisms, the mTOR pathway plays additional roles, including cell-cycle regulation and maintenance of cell survival, and it is therefore an attractive target for novel anticancer agents [19,23,24]. This is supported by preclinical data using rapamycin and its analogues, which suggest a broad range of anticancer activity, and also by early clinical trial data, including evidence for activity in acute myeloid leukemia (AML) patients [25]. It is, therefore, relevant to ask if mTOR and its targets are constitutively activated in cancer patients, and also to determine the extent to which this is further enhanced by inputs from the ERK pathway.
Materials and methods Patients These were newly diagnosed acute leukemia patients being treated at the Princess Margaret Hospital, Toronto. Following preliminary work to optimize the flow cytometry technique, a total of 19 patients were included in the present series. These were selected based on the presence of circulating blast cells. According to institutional research ethics guidelines, 5 mL peripheral blood was obtained, using heparin as the anticoagulant, and processed within 24 hours in the flow cytometry laboratory. In addition to the leukemia patient samples, we also examined granulocyte colony-
1183
stimulating factor (G-CSF) mobilized CD34þ blasts in peripheral blood samples obtained from allogeneic stem cell transplant donors. Reagents and antibodies Wash buffer consisted of calcium- and magnesium-free phosphate-buffered saline containing 4% fetal bovine serum. Phorbol myristate acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) was diluted to a 40 mM working solution from a 40 mM stock in ethanol. Stem cell factor (SCF) was obtained from Calbiochem (San Diego, CA, USA), and diluted to 10 mg/mL in wash buffer. Insulin-like growth factor-1 (IGF-1) was obtained from PeproTech (Rocky Hill, NJ, USA) and made up to 10 mg/mL in wash buffer. The mitogen-activated protein kinase kinase (MEK) inhibitor U0126 (Cell Signaling Technology, Beverly, MA, USA) was prepared as a 10-mM solution in methanol. Rapamycin (Calbiochem) was diluted to a 100 mg/mL working solution from a 1 mg/mL stock in dimethylsulfoxide. LY294002 (Calbiochem) was prepared as a 50-mM solution in ethanol. A rabbit polyclonal antibody to pS6 Ser 235/236 (#2211), rabbit monoclonal antibody to pAkt Ser 473 (#4058), and a mouse monoclonal anti-phosphorylation of extracellular signal-regulated kinase (pERK) 1/2 (Thr 202/Tyr 204; #9106) conjugated to Alexa Fluor 488 were obtained from Cell Signaling Technology. Surface markers CD34-phycoerythrin (PE) (clone 581) and CD45-PE (clone J.33) were obtained from Beckman Coulter, Inc. (Miami, FL, USA). Alexa Fluor 647-conjugated goat anti-rabbit-IgG F(ab0 )2 was purchased from Molecular Probes (Eugene, OR, USA). Flow cytometry protocol Figure 1 gives a schematic of the interactions between mTOR and ERK that are relevant to this work. The flow cytometry protocol analyzed these interactions by measuring the basal levels of pS6 and pERK in the leukemic blast population, and how these alter in response to agents that inhibit upstream signaling elements, as well as to ex vivo activation via c-Kit using SCF. Whole blood samples were divided into 200 mL aliquots in 5 mL round-bottomed polypropylene tubes, and placed in a 37 C dry bath. To one set of tubes, inhibitors (100 mM U0126, 1 mg/mL Rapamycin, or 500 mM LY294002) were added and incubated for 30 minutes. A second set of tubes was pretreated with inhibitors for 25 minutes, and then activated by adding SCF 100 ng/mL for 5 minutes. As a positive control for ERK activation, 400 nM PMA was added to an additional sample for 10 minutes. Previous work from our laboratory ([7] and unpublished data) has found that PMA strongly activates ERK in all nucleated blood cells, regardless of whether these are able to respond to SCF stimulation. Following treatment, samples were processed using a protocol for fixation, red cell lysis, and permeabilization that was recently developed in our laboratory, and optimizes the preservation of phenotypic features as well as intracellular phosphorylated epitopes in whole blood samples [26]. Briefly, samples were removed from the dry bath, and fixed by adding methanol-free formaldehyde (10% solution; Polysciences, Warrington, PA, USA) to give a final concentration of 4% for 10 minutes. Then 2 mL 0.1165% Triton X-100 (final concentration 0.1%) was added to lyse red blood cells. After additional incubation for 30 minutes at room temperature, 2 mL cold wash buffer was added. Cells were centrifuged for 3 minutes at 1000g at 10 –15 C. The
1184
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
Figure 1. Schematic showing major pathways and sites of action of agents used in this study. eIF-4E 5 eukaryotic initiation factor-4E; ERK 5 extracellular signal-regulated kinase; MEK 5 mitogen-activated protein kinase kinase; mTOR 5 mammalian target of rapamycin; PI3 5 phosphatidylinositol 3; SCF 5 stem cell factor.
supernatant was aspirated, and the cell pellet resuspended in 1 mL cold freezing medium consisting of 10% glycerol, 20% fetal bovine serum in RPMI 1640 tissue culture medium, and stored at 20 C until antibody staining.
525, 575, and 675 nM. Fluorescence compensation was not used. Listmode analysis was done with FlowJo software (Tree Star, Inc., Ashland, OR, USA).
Antibody staining As soon as frozen samples were thawed, aliquots containing 0.5 million cells were diluted into 2 mL cold wash buffer then centrifuged. Cells were resuspended in 1 mL 50% ice-cold methanol in 0.9% NaCl, and held on ice for 10 minutes. After removal of methanol, cells were washed with 2 mL wash buffer, and the cell pellet resuspended and labeled with either 0.5 mL unconjugated anti-pS6 Ser 235/236 or 1 mL unconjugated anti-Akt Ser 473 for 15 minutes at room temperature. Samples were washed and then stained with an antibody cocktail consisting of 10 mL Alexa Fluor 488-conjugated anti-pERK, the Alexa Fluor 647conjugated secondary antibody, and PE-conjugated anti-CD34. In acute leukemia cases, that were CD34, the combination of CD45-PE staining intensity and orthogonal light scatter was substituted in order to identify leukemic blasts [27]. After 15 minutes incubation at room temperature, samples were washed and resuspended in 150 mL wash buffer for flow cytometry.
Results and discussion
Flow cytometry This was done using a modified Epics Elite cell sorter (Beckman Coulter, Inc., Miami, FL, USA), with spatially separated aircooled argon and HeNe lasers, each emitting at 20 mW. Fluorescence signals were collected through bandpass filters centered at
Activation of S6 in mobilized peripheral stem cells from normal donors Mobilized peripheral stem cells were included in the study in order to gain insight into S6 activation pathways in genotypically normal CD34þ blasts in peripheral blood samples. A total of four cases were examined; the results were very similar in all of these. As shown in Figure 2, the sample preparation protocol gave excellent preservation of CD34 staining and orthogonal light scatter, allowing unambiguous identification of the blast cell population. Bivariate plots of pERK vs pS6 showed that fluorescence intensities for the blast cells were within a narrow range of values. There was no discernable decrease in either pERK or pS6 following preincubation with rapamycin, LY294002, or U0126 (data not shown), suggesting that the fluorescence signals represent background due to autofluorescence and nonspecific antibody binding. Treatment with SCF resulted in an increase in both pERK and pS6 in the blast population, as illustrated in Figure 2. In
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
1185
Figure 2. Monitoring signaling pathways in granulocyte colony-stimulating factor–mobilized peripheral stem cells. The sample preparation technique readily identifies these, based on light scatter and CD34 surface staining (top panels). Lower left panels show bivariate dot plots of pS6 vs phosphorylation of extracellular signal-regulated kinase (ERK), and responses to the indicated treatments. Note that both stem cell factor (SCF) and phorbol myristate acetate (PMA) activate ERK and S6, whereas insulin-like growth factor-1 (IGF-1) has no significant effect. The effects of SCF and PMA (not shown) on ERK and S6 are inhibited by U0126 but not by rapamycin, indicating that acute activation of S6 is predominantly mediated by the ERK pathway. Bottom right panels show Ser473 Akt phosphorylation on the vertical axis. Note that whereas SCF activates both ERK and Akt, PMA activates ERK but not Akt, and IGF-1 activates Akt but not ERK. Quadrants define labeling intensities of the unstimulated control samples, and are placed to aid visual inspection.
contrast, stimulation using IGF-1 resulted in activation of Akt with minimal effects of ERK, and did not phosphorylate S6 (Fig. 2). Treatment with PMA activated pERK and pS6 but not pAkt in CD34þ blasts, as well as in the lymphocyte, neutrophil, and monocyte populations. Pretreatment with U0126 inhibited activation of both pERK and pS6 by SCF and PMA, whereas rapamycin was ineffective. Taken together, these results indicate low or absent basal levels of ERK and S6 phosphorylation in mobilized peripheral stem cells. When these cells are acutely activated by SCF, ERK rather than mTOR appears to be the predominant pathway for serine 235/236 phosphorylation of S6.
Constitutive S6 phosphorylation in the peripheral blasts of acute leukemia patients Table 1 gives a summary of the patient demographics, including the type of acute leukemia, cytogenetic analysis, and clinical outcome. Six patients received either no chemotherapy or low-dose palliative treatment because of age or other medical conditions. Of those treated with intensive chemotherapy, 10 achieved hematological complete remission, and the remainder showed gross evidence of residual disease in the bone marrow after chemotherapy. Patients were selected for study based on the presence of at least 5% circulating blast cells. However, because of the excellent preservation of light scatter and surface
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
1186 Table 1. Patient information
Patient no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Type
Cytogenetics
Age (y)
Increased basal pS6
Rapamycinsensitive
LY294002sensitive
U0126sensitive
SCFresponse
Clinical outcome
AML (M2) ALL (Ph-ve) MDS/AML AML (M2) AML (M2) MDS/AML AML (M2) ALL (Ph-ve) AML (M4) AML (M7) AML (M0) MDS/AML AML (M2) AML (M2) PRV/AML AML (M4) ALL (Phþve) APL (M3) AML (M2)
46XY, inv(20) (q12q31.1)
38 42 72 44 72 67 25 47 26 46 80 76 77 79 62 48 74 45 18
þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ
þ þ þ þ þ þ
þ þ þ þ
þ
þ
þ þ þ þ
þ þ þ þ
þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
CR CR Palliative CR Palliative Palliative CR Palliative CR Progressed Palliative Palliative Progressed CR CR CR CR CR Progressed
47XY, þ13 46XX (Flt3 ITD) 46XY add (6p) 45XY, -7 46XX, t(8;21) 46XY, t(8;21) 46XY 46XY 46XX 46XX 46XY 46XY 46XX, t(9;22) 46XX, t(15;17) 4XX
ALL 5 acute lymphocytic leukemia; AML 5 acute myeloid leukemia; APL 5 acute promyelocytic leukemia; CR 5 complete response; MDS 5 myelodysplastic syndrome; PRV 5 polycythemia rubra vera; SCF 5 stem cell factor.
immunophenotype achieved by the flow cytometry protocol, we found that blast populations that were !1% of the total white blood cell count could be readily identified. Figure 3 illustrates examples of the patterns of signaling pathway activation, as well as the gating strategy based on light scatter and surface immunophenotype used to identify the blast population. In contrast to mobilized peripheral stem cells, rapamycin, LY294002, and U0126 produced significant decreases in S6 phosphorylation in the blast population, as summarized in Tables 1 and 2. Typically S6 phosphorylation showed considerable cellular heterogeneity within individual patients. For example, patient no. 6 (Fig. 3) shows a range in basal levels of pS6 of more than 100-fold. With one exception, rapamycin suppressed pS6 levels in those samples showing constitutive activation, as a result of which, the range of individual cell values became much narrower. Taking into account the often extreme cellular heterogeneity and responses to inhibitors, we categorized 11 of 19 samples as showing elevated pS6 levels (Table 1). Because rapamycin is a highly specific inhibitor of mTOR, these results suggest that the mTOR pathway is active in approximately half of acute leukemia patients. The phosphatidylinositol 3 (PI3)-kinase/Akt pathway is believed to be an important upstream activator of mTOR. Although Akt can directly phosphorylate mTOR, the significance of this is not certain, and mTOR activation appears to occur by a more circuitous route involving other signaling elements including tuberin (TSC2) and the small GTP-binding protein Rheb [18,28,29]. In preliminary experiments using a rabbit monoclonal antibody to Akt
serine 473, we found increased levels of expression in some samples showing elevated S6 phosphorylation, suggesting that constitutive Akt activation is involved in the rapamycin-sensitive phosphorylation of S6 in some acute leukemia patients (Fig. 4). This is further supported by the inhibitory effects on pS6 after treatment with the PI3-kinase inhibitor LY294002, although it should be noted that LY294002 and the structurally unrelated PI3-kinase inhibitor wortmannin both have been reported to inhibit mTOR [30]. Therefore, we cannot exclude the possibility that the effects of LY294002 on pS6 could be due to mTOR rather than to PI3-kinase inhibition. Effects of U0126 Constitutive pERK has been reported to occur in some AML patients [12,31]. However, in contrast to the extensive cellular heterogeneity seen with pS6, baseline pERK levels in the peripheral blast populations were narrowly distributed, and in all cases showed mean values that were only slightly greater than background autofluorescence. Furthermore, as summarized in Table 3, although there was a small decrease in the mean level of pERK in samples pretreated with U0126 (0.63 0.41 vs 0.51 0.40 arbitrary units), this was not statistically significant. Nevertheless, treatment with U0126 resulted in a significant decrease in the mean pS6 levels in the series of 19 samples as shown in Tables 1 and 2. By visual inspection of the pERK vs pS6 bivariate dot plots this was evident in 6 of the 11 samples that contained high pS6 cells (Table 1, Fig. 3). It should be noted that the concentration of U0126 used in these experiments was approximately 10-fold greater than that typically used
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
1187
Figure 3. Bivariate plots of pS6 vs phosphorylation of extracellular signal-regulated kinase (pERK) obtained from six acute myeloid leukemia (AML) patients, illustrating the effects of treatment with rapamycin, LY294002, and U0126 vs untreated control. Left panels show the respective plots of orthogonal light scatter vs CD34 or CD45, and illustrate the gating regions used to identify the blast population. Patient identifier is given in the lower right corner of these plots. Note extreme heterogeneity seen in pS6 levels in patient nos. 3, 6, and 14. This is strongly inhibited by rapamycin and LY294002, and to a lesser extent by U0126. PE 5 phycoerythrin.
to inhibit MEK in vitro, but this was based on our previous experience and is needed because of greater protein binding of U0126 in whole blood [7]. Because U0126 is considered to be a selective MEK inhibitor, we suggest that the effects on S6 phosphorylation in acute leukemia patient samples indicate the presence of low levels of constitutive pERK activity that, in concert with mTOR signaling, are able to enhance S6 phosphorylation. This effect is clearly seen in Figure 5 (patient no. 5), where the blast population defined by CD34 and side scatter
shows greater inhibition of pS6 with U0126 compared with rapamycin, and is even more pronounced in a CD34/high side-scatter subpopulation, where the cells with the greatest levels of pS6 also show high levels of pERK that are inhibited by U0126. Effects of SCF on AML blasts Table 3 summarizes the effects of stimulation with SCF and PMA on mean pERK levels. In 15 of 19 patients ex vivo treatment with SCF produced acute activation of signaling
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
1188
Table 2. Mean P-S6 values for blast populations
Table 3. Mean pERK values for blast populations
Untreated
Rapamycin
LY294002
U0126
15.0 11.5
7.8 4.3*
7.6 4.3**
9.7 6.1***
*p 5 0.006; **p 5 0.007; ***p 5 0.013; two-tailed paired t-test; comparing to untreated control.
pathways and, in 14 cases, this resulted in increases in both ERK and S6 phosphorylation, similar to the example shown in Figure 5. In the remaining case, there was no effect on ERK or S6, but there was a large increase in Akt phosphorylation. As shown in Table 1, all of the AML cases were responsive to SCF, whereas stimulation was not seen in any of the three cases of acute lymphocytic leukemia or the single case of acute promyelocytic leukemia. Similar to results obtained with mobilized peripheral stem cells, acute activation of S6 after SCF treatment was inhibited by U0126 but not rapamycin; this was also seen with PMA activation. In contrast to the constitutive S6 phosphorylation seen in AML blasts, where mTOR signaling predominates, acute activation via SCF occurs through the ERK pathway. Conclusions Overall, the results described here support the recent report by Irish et al. [8] that flow cytometry is able to reveal signaling complexity in AML patient samples. In this article, we used a recently developed whole-blood fixation technique that allows measurement of constitutive phosphoryla-
Untreated 0.63 0.41
U0126
SCF
PMA
0.51 0.4
2.54 3.23
7.94 3.35
pERK 5 phosphorylation of extracellular signal-regulated kinase; PMA 5 phorbol myristate acetate; SCF 5 stem cell factor.
tion states, as well as the response to ex vivo treatment with growth factors and inhibitors of signaling pathways [26]. This allowed identification of high levels of constitutive phosphorylation of the S6 ribosomal protein in more than half of AML patients tested, as well as revealing a complex interaction between the Akt/mTOR and ERK signaling pathways that appears to show considerable inter-patient heterogeneity. These effects were not seen in mobilized peripheral stem cells from normal donors, and most likely reflect alterations in upstream signaling elements, such as N-ras or flt-3 that occur as part of the leukemic process. Based on the overall greater effect of rapamycin compared with U0126, we propose that in most cases of AML, mTOR is the predominant mediator of constitutive S6 phosphorylation. However, in the example shown in Figure 5, S6 phosphorylation is driven by ERK signaling, and this case is relatively refractory to rapamycin, suggesting that AML patients might be stratified based on constitutive activation of the upstream pathways. This would have implications for selection of drugs targeting different signaling molecules. In contrast to the predominance of mTOR in constitutive
Figure 4. Dual labeling of pAkt (S473) and phosphorylation of extracellular signal-regulated kinase (pERK) in patient no. 3 (top panels), compared with pS6 vs pERK (lower panels). Similar to pS6, pAkt shows a heterogeneous distribution in the untreated control. Preincubation with LY294002 results in decreased pAkt, whereas stem cell factor (SCF) acutely activates pAkt as well as pERK in all of the CD34þ blasts.
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
1189
Figure 5. Constitutive of extracellular signal-regulated kinase (ERK) activation of S6 in a subpopulation defined by increased orthogonal light scatter and loss of CD34 expression. As seen in the lower right panel, this population is responsive to stem cell factor (SCF), indicating that it is likely of leukemic origin. Note the great heterogeneity of pS6 in these cells, with clear evidence for increased phosphorylation ERK (pERK) in cells at the upper extreme of pS6. In this example U0126 strongly inhibits pS6 as expected, whereas rapamycin has minimal effects. This pattern is also seen in the CD34þ population, illustrated in the upper set of panels. PE 5 phycoerythrin.
S6 phosphorylation, ERK predominates when S6 is activated by ex vivo treatment with SCF; similar to the effects seen with the mobilized normal peripheral stem cells. Given that the Akt/mTOR pathway appears to play a significant role in cell survival signaling, it will be of interest to determine if high constitutive levels are predictive of acute leukemia patient outcome, although this was not evident in the present small series, as shown in Table 1. Our original motivation for developing whole-blood flow cytometry methods to study signaling pathways was the need for pharmacodynamic monitoring of novel agents, where isolation techniques are likely to perturb the drug/target equilibrium [7]. The method described in the present ar-
ticle appears to be well-suited to meet this need, and has the potential to interrogate several signaling elements of potential importance in AML, such as c-kit, flt-3, PI3-kinase, as well as mTOR. It should also be noted that relative to previously described techniques from our own laboratory and from others, standard phenotypic features of light scatter and surface immunophenotype are well preserved in clinical samples, offering the potential to extend applications to rare events, including study of minimal residual disease. Acknowledgments The authors thank Dr. James Jacobberger, Case Western Reserve University; Dr. T. Vincent Shankey, Advanced Technology Center,
1190
S. Chow et al./ Experimental Hematology 34 (2006) 1182–1190
Beckman-Coulter; and Dr. Bradley Smith, Cell Signaling Technology, for their critical comments. This work was supported by a grant from the Ontario Cancer Research Network and research collaboration support was provided by Advanced Technology Center, Beckman Coulter, Inc., Miami FL, USA.
References 1. Bernasconi P, Boni M, Cavigliano PM, et al. Molecularly targeted therapy in acute myeloid leukemia. Ann N Y Acad Sci. 2004;1028: 409–422. 2. Levis M. Recent advances in the development of small-molecule inhibitors for the treatment of acute myeloid leukemia. Curr Opin Hematol. 2005;12:55–61. 3. Sattler M, Scheijen B, Weisberg E, Griffin JD. Mutated tyrosine kinases as therapeutic targets in myeloid leukemias. Adv Exp Med Biol. 2003;532:121–140. 4. Sawyers C. Targeted cancer therapy. Nature. 2004;432(7015):294– 297. 5. Stirewalt DL, Meshinchi S, Radich JP. Molecular targets in acute myelogenous leukemia. Blood Rev. 2003;17:15–23. 6. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001;344:1031–1037. 7. Chow S, Patel H, Hedley DW. Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry. 2001;46(2):72–78. 8. Irish JM, Hovland R, Krutzik PO, et al. Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell. 2004;118:217–228. 9. Jacobberger JW, Sramkoski RM, Frisa PS, et al. Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity. Cytometry. 2003;54A(2):75–88. 10. Perez OD, Nolan GP. Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat Biotechnol. 2002;20:155–162. 11. Pallis M, Seedhouse C, Grundy M, Russell N. Flow cytometric measurement of phosphorylated STAT5 in AML: lack of specific association with FLT3 internal tandem duplications. Leuk Res. 2003;27:803–805. 12. Ricciardi MR, McQueen T, Chism D, et al. Quantitative single cell determination of ERK phosphorylation and regulation in relapsed and refractory primary acute myeloid leukemia. Leukemia. 2005;19: 1543–1549. 13. Tong FK, Chow S, Hedley DW. Pharmacodynamic monitoring of BAY 43-9006 (Sorafenib) in phase i clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells. Cytometry B Clin Cytom 2006;70:107–114. 14. Herbert TP, Tee AR, Proud CG. The extracellular signal-regulated kinase pathway regulates the phosphorylation of 4E-BP1 at multiple sites. J Biol Chem. 2002;277:11591–11596. 15. Wang L, Proud CG. Ras/Erk signaling is essential for activation of protein synthesis by Gq protein-coupled receptor agonists in adult cardiomyocytes. Circ Res. 2002;91:821–829.
16. Abraham RT. mTOR as a positive regulator of tumor cell responses to hypoxia. Curr Top Microbiol Immunol. 2004;279:299–319. 17. Alvarez B, Garrido E, Garcia-Sanz JA, Carrera AC. Phosphoinositide 3-kinase activation regulates cell division time by coordinated control of cell mass and cell cycle progression rate. J Biol Chem. 2003;278: 26466–26473. 18. Brugarolas J, Lei K, Hurley RL, Manning BD, et al. Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 2004;18:2893–2904. 19. Wendel HG, De Stanchina E, Fridman JS, et al. Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature. 2004; 428(6980):332–337. 20. Gingras AC, Gygi SP, Raught B, et al. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev. 1999;13: 1422–1437. 21. Fingar DC, Richardson CJ, Tee AR, Cheatham L, Tsou C, Blenis J. mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E. Mol Cell Biol. 2004;24:200–216. 22. Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell. 2005;121:179–193. 23. Mita MM, Mita A, Rowinsky EK. The molecular target of rapamycin (mTOR) as a therapeutic target against cancer. Cancer Biol Ther. 2003;2(suppl 1):S169–S177. 24. Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute myeloid leukemia cells requires PI3 kinase activation. Blood. 2003; 102:972–980. 25. Recher C, Beyne-Rauzy O, Demur C, et al. Antileukemic activity of rapamycin in acute myeloid leukemia. Blood. 2005;105:2527–2534. 26. Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV. Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A. 2005;67:4–17. 27. Borowitz MJ, Guenther KL, Shults KE, Stelzer GT. Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis. Am J Clin Pathol. 1993;100:534–540. 28. Garami A, Zwartkruis FJ, Nobukuni T, et al. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol Cell. 2003;11:1457–1466. 29. Nave BT, Ouwens M, Withers DJ, Alessi DR, Shepherd PR. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J. 1999; 344(pt 2):427–431. 30. Brunn GJ, Williams J, Sabers C, Wiederrecht G, Lawrence JC Jr, Abraham RT. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. 1996;15:5256–5267. 31. Kim SC, Hahn JS, Min YH, Yoo NC, Ko YW, Lee WJ. Constitutive activation of extracellular signal-regulated kinase in human acute leukemias: combined role of activation of MEK, hyperexpression of extracellular signal-regulated kinase, and downregulation of a phosphatase, PAC1. Blood. 1999;93:3893–3899.