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p70S6 kinase phosphorylation for pharmacodynamic monitoring
Clinica Chimica Acta 413 (2012) 1387–1390
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Invited critical review
p70S6 kinase phosphorylation for pharmacodynamic monitoring B. Hartmann ⁎ Zentrum für Innere Medizin, Klinik für Innere Medizin I, Sektion Nephrologie, Albert Einstein Allee 23, D-89070 Ulm, Germany
a r t i c l e
i n f o
Article history: Received 29 April 2011 Received in revised form 21 March 2012 Accepted 26 March 2012 Available online 13 April 2012 Keywords: mTOR inhibitor Rapamycin Sirolimus Everolimus Pharmacodynamic monitoring Kidney transplantation
a b s t r a c t Inhibitors of the mammalian target of rapamycin (mTOR) are administered as immunosuppressant as well as antineoplastic agents. Because of the narrow therapeutic index of mTOR inhibitors, drug monitoring is required, and this is usually done by measuring blood drug levels. Increasing knowledge of the signaling pathways of the mTOR protein kinase provides an opportunity for pharmacodynamic drug monitoring. With the different laboratory methods it is becoming possible to measure new biomarkers to control the influence of mTOR activity. One of these biomarkers is phospho-S6 kinase, with its isoform p70S6K. © 2012 Published by Elsevier B.V.
Contents
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1390
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase. It plays a central role in a great many cellular functions including metabolism, growth, survival, aging, synaptic plasticity and memory [1]. This signaling serine/threonine kinase is divided into two distinct kinase complexes, namely mTORC1 and mTORC2 [2]. The mTORC1 phosphorylates S6 kinase 1 with its isoforms p70S6 kinase (p70S6K) and p85S6K. The mTORC2 is rapamycin insensitive. It regulates phosphorylation of Akt, PKCα (protein kinase Cα) and SGK1 (serum- and glucocorticoid-induced protein kinase 1) and is thought to regulate actin cytoskeleton dynamics. mTOR inhibitors, namely sirolimus (rapamycin) and its derivative everolimus, target mTORC1. Rapamycin is a macrocyclic antibiotic, first described in 1975, produced by Streptomyces hygroscopicus. It was later renamed sirolimus because of its structural similarity to tacrolimus and because it was found to have immunosuppressive and antiproliferative properties [3]. Sirolimus and its derivative, everolimus, bind to the FK binding protein and inhibit mTORC1 mediated signal transduction pathways [2,4,5] (Fig. 1). mTORC1 controls the hydrophobic motif of p70 ribosomal S6
⁎ Tel.: + 49 731 500 44714; fax: + 49 731 500 44567. E-mail address: [email protected]. 0009-8981/$ – see front matter © 2012 Published by Elsevier B.V. doi:10.1016/j.cca.2012.03.023
kinase [4,5]. Two isoforms of S6K1 are produced from the same transcript by alternative initiation of translational start sites: the shorter form of S6K1, which is largely localized in the cytoplasm, is termed p70S6K. A second isoform, p85S6 kinase, is derived from the same gene and is identical to p70S6 kinase except for 23 extra residues at the amino terminus, which encode a nuclear localizing signal [6]. The functional significance of the differential subcellular localization of the two S6K1 isoforms has not been established, although it is tempting to speculate that the nuclear form is involved in phosphorylation of the nuclear pool of the free, chromatin-bound form of S6 [7]. p70S6K is probably one of the best characterized downstream effectors of mTORC1 [8]. In the late 1990s, clinical trials confirmed the efficacy of sirolimus as a potent immunosuppressant in renal transplantation without it appearing to cause a significant inherent nephrotoxicity [9]. Apart from immunosuppression in renal transplantation as an alternative to nephrotoxic calcineurin inhibitors, mTOR inhibitors are increasingly used in heart and liver transplantation. mTOR is also a key target for therapeutic intervention in cancer, as an important regulator of cell growth [10]. mTOR inhibitors are used, for example, for chemotherapy of soft tissue and bone sarcomas [11]. Sirolimus and everolimus are critical dose drugs with a low therapeutic index. Under or overdosing can lead respectively to rejection or toxicity. mTOR inhibitors are associated with a number of possible
1388
B. Hartmann / Clinica Chimica Acta 413 (2012) 1387–1390
A simplyfied model of the mTOR pathway Cellular stress, Cellular nutrition, Insulin, ILGF, etc.
Sirolimus or Everolimus
+ Sirolimus or Everolimus
-
+
mTORC1
FKBP
-
mTORC2
FKBP
P
P
4E-BP1
Protein synthesis
p70S6K
Cell growth
Cell proliferation
Fig. 1. A simplified model of the mTOR pathway: mTOR has a central role in cell metabolism and is found in two distinct protein complexes (mTORC1 and mTORC2). Only mTORC1 is sensitive to rapamycin (i.e. sirolimus and everolimus). Active mTORC1 phosphorylates its downstream substrates 4E-BP1 and p70S6K, thus stimulating protein synthesis and cell growth. mTOR inhibitors such as sirolimus and everolimus bind to the FK binding protein 12 (FKBP12) and, as this complex, inhibit mTORC1 and its downstream effects. Adapted from [31].
adverse effects, including leukopenia, thrombocytopenia, anemia, hypercholesterolemia, hypertriglyceridemia and diarrhea, as well as wound healing problems, interstitial pneumonitis and proteinuria. Although there are many benefits of using mTOR inhibitors in renal transplantation, approximately 30–50% of patients on mTOR inhibitor therapy discontinue it during follow-up due to the related side effects [12–15]. Adjustment of mTOR inhibitor doses currently relies entirely on sirolimus or everolimus blood concentrations, which may not necessarily correlate with the pharmacological effects of the drugs on immune cells. Recommended trough levels for sirolimus, derived from a ten-year perspective, are at present from 5 to 15 ng/ml, which is too wide a range for reliable immunosuppressive therapy. Knowledge of the pharmacodynamic processes of immunosuppressive agents offers the opportunity of estimating the clinical effect and thereby of further refining and optimizing current therapy [16]. The phosphorylation of p70S6K as a well characterized downstream effect of mTOR makes measuring the activity of this enzyme a suitable target for pharmacodynamic monitoring of mTOR inhibition [17,18]. Studies of rapamycin-sensitive regulatory phosphorylation sites showed that rapamycin-sensitive sites were phosphorylated in response to mitogenic stimulation [19] and that the three sites underwent hierarchical dephosphorylation by rapamycin treatment, with T389 > S404 > T229, T389 dephosphorylation most closely paralleling loss of kinase activity [20]. Conversion of these sites to either acidic or neutral amino acids revealed that T229 and T389 were critical regulatory sites, whereas S404 appeared to play a modulatory role. Mutational analysis also revealed that the principal site of rapamycin inducing S6K1 inactivation was T389, and substitution of an acidic residue in this position resulted in a kinase variant which increased basal activity and was largely rapamycin resistant [21]. Different approaches have already been attempted to establish the usefulness of pharmacodynamic monitoring of mTOR inhibition on the basis of measuring p70S6K activity. This was estimated in white blood cells after treatment with rapamycin using a radioactivelabeled phospho-p70S6K antiserum. Although assay robustness was poor, it was possible to show a difference between lower and higher mTOR inhibition, as well as lack of influence on cells treated with calcineurin inhibitors in vitro by measuring the counts per min (cpm) [17].
The role of p70S6K activity in promoting the growth of small cell lung cancer (SCLC) was evaluated in cell growth experiments, in colony formation assays and by measuring p70S6K activity with Western blot analysis after treatment of SCLC cell lines with different concentrations of rapamycin. Cell growth, colony forming factor and phosphorylation status of p70S6K were reduced with increasing sirolimus concentrations [22]. In 2003, Peralba et al. [23] reported that the treatment with CCI-779, an ester of sirolimus, of nude mice bearing the CCI-779 susceptible breast cancer cell line MDA-468 resulted in high inhibition of p70S6K activity in peripheral blood mononuclear cells (PBMC). Furthermore, the degree of inhibition was identical in PBMC and simultaneously collected tumor tissue, suggesting that the PBMCs were an adequate surrogate tissue for p70S6K activity in vivo. The activity of the p70S6K was measured by Western blot analysis. Sirolimus based immunosuppressive therapy in renal transplant patients was monitored in a small study for the first time by measuring the trough level of sirolimus and by assessing the phosphorylation status at the Thr389 site of p70S6K in PBMCs using Western blot [24]. A total of 36 patients with renal transplants and 8 healthy controls were enrolled. Results suggested that sirolimus treatment was associated with pronounced inhibition of p70S6K phosphorylation, compared with results from healthy controls or patients treated with other immunosuppressant therapy. Remarkably, no correlation of the sirolimus trough level with reduction in phospho-p70S6K could be demonstrated. Furthermore, some patients exhibited low trough concentrations yet a strong reduction in phospho-p70S6K, indicating individual enzyme susceptibility. There were five episodes of clinically relevant rejection during the study period. All the patients who experienced acute rejection uniformly maintained a high degree of enzyme phosphorylation, whereas non-rejecters showed significant inhibition of phosphorylation. A reduction in p70S6K phosphorylation status below 60% in comparison with the results from healthy controls was considered as significant in that survey. In all patients with a trough level equal or above 6 ng/mL, p70S6K phosphorylation was constantly inhibited. This study concluded that the phosphorylation status of p70S6K appeared to provide more relevant information on the desired effect of sirolimus in target cells than trough concentration measurements.
B. Hartmann / Clinica Chimica Acta 413 (2012) 1387–1390
Apart from their capacity for immunosuppression, mTOR inhibitors are being increasingly recognized as an anticancer therapy. Cancer is also a side effect of long-term immunosuppression. The prevalence of some cancers, especially skin cancer, is increased in transplanted patients, so it is important to know which cancer will be sensitive to rapamycin treatment. The pharmacodynamic approach implies the need for biochemical monitoring of immunosuppressive therapy based on mTOR inhibitors, as well as identification of increased mTOR activity due to growth of the cancer. In a study with renal transplant recipients with Kaposi sarcoma, markedly increased mTOR activity was shown by measuring increased p70S6K activity by Western blot analysis [25]. p70S6K phosphorylation was estimated in PBMCs from 37 patients being treated with a cyclosporine-based immunosuppressive therapy, of whom ten had Kaposi sarcoma. Six months after switching to rapamycin therapy, p70S6K activity was substantially reduced in both groups. This reduction was associated with regression of the dermal neoplasm in the patients with Kaposi sarcoma. Over-activation of basal p70S6K in PBMCs from renal transplant recipients thus appeared to be associated with Kaposi sarcoma dermal lesions, and inhibition of p70S6K was linked to regression of skin cancer lesions. The influence of co-medication with tacrolimus on the potency of sirolimus for reducing phosphorylation of p70S6K was investigated in another study with kidney transplant patients [26]. The activity of p70S6K in PBMCs was also measured by Western blotting and the results were compared with the sirolimus trough level measured in parallel. From this survey it could be concluded that co-medication with tacrolimus limited the inhibitory effect of sirolimus on p70S6K activation and – as had been shown by others before – there is no correlation between p70S6K phosphorylation and trough levels, because of high individual variation in p70S6K phosphorylation. In all the studies mentioned above, Western blot analysis was used to measure the activity of p70S6K. PBMCs were isolated from a freshly drawn blood by means of Ficoll-Hypaque gradient centrifugation. The procedure was performed in the presence of protease and phosphatase inhibitor cocktails. The cells were then lysed and subjected to blotting as described in the literature. Monoclonal antiphospho-p70S6K antibodies were applied directed against the phosphorylation sites serine 411 or threonine 389. In some studies, p70S6K generation was stimulated in vivo as well as in vitro by growth factors such as insulin. The different procedures to analyze the time from drawing to lysing the blood were not described. Nor was there any information about inter- and intraassay variation. The Western blot technique has the advantage of not being very expensive and it is an established test in any laboratory. Protein immunoblot analysis is however not a quantitative technique and is not a suitable test for large population studies and inter-subject comparisons. As an alternative to p70S6K measurement there is a possibility to measure the mTOR activity itself. There is an ELISA-based assay for measuring the kinase activity of mTOR. This test can be applied for in vitro mTOR inhibitor screening and for assessing the regulation of mTOR cell signaling. With this method mTOR activity was studied in kidney transplant recipients in one study [27]. PBMC samples from five renal allograft patients were obtained before treatment with everolimus and one month after the start of everolimus therapy. With this approach, no significant difference in mTOR activity could be seen between the two groups. The conclusion from these preliminary results is that there is no advantage in directly measuring mTOR activity for measuring p70S6K activity in order to monitor the biochemical effect of mTOR inhibitor therapy. Two other assays in particular, FACS analysis and ELISA, are presently being studied for analyzing p70S6K activity. The results of phosphorylated S6 ribosomal protein (p-S6RP) measurement by FACS analysis have recently been published. Phospho-specific flow cytometry, or phospho flow, measures the phosphorylation status of intracellular proteins at the single cell
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level [28]. In one study to evaluate the eligibility of this analytical procedure, blood from five volunteers was analyzed. The blood was incubated with different clinically relevant concentrations of either sirolimus or other immunosuppressive drugs, e.g. cyclosporine A, mycophenolic acid or dexamethasone. Following drug treatment, the whole blood samples were stimulated by adding phorbol 12-myristate 13-acetate (PMA) and ionomycin, in order to increase the amount of p-S6RP. After this procedure the whole blood was analyzed by flow cytometry to measure p-S6RP in T-cells. As expected, treatment with sirolimus showed suppression of p-S6RP. Neither cyclosporine A, mycophenolic acid nor dexamethasone had any effect on mTOR-related p-S6RP. In this small survey with blood samples from five volunteers, intra and interassay, as well as interindividual variability were examined. The results were 3 to 5%, 12 to 25% and 14 to 38%, respectively. Furthermore, the time from blood collection to analyzing it was measured under different conditions (room temperature, 4 °C). It could been shown that samples can be stored at RT or 4 °C for up to 2 h after withdrawal [29]. Blood samples from patients with acute myelogenous leukemia (AML) were analyzed with the same technique to measure p-S6RP. The mTOR signaling pathway is frequently activated in AML, but it has been stated that S6 phosphorylation in AML blasts is heterogeneous and, in some cases, intrinsically resistant to rapamycin at clinical concentrations. The mTOR inhibitor sirolimus was added to chemotherapy with mitoxantrone, etoposide and cytarabine. Inhibition of p-S6RP in the majority of subjects' tumor cells could be shown during therapy using a whole blood fixation/permeabilization technique for flow cytometry [30]. According to the results of the two studies, it seems that phospho-specific flow cytometry is a reliable test for measuring p-S6RP in T-cells. Its advantage is that the target cells (T-lymphocytes) can be measured directly, since the test allows the phosphorylation status of intracellular proteins to be measured at the single cell level. ELISA based measurement of p70SK is also possible. In a cell culture model (Jurkat cells and PBMCs from buffy coats), the phosphorylation status was analyzed of p70S6K. Following treatment of the cells with sirolimus, they were stimulated with the phorbol ester PMA. Phospho-p70S6K expression was measured by (i) semiquantitative Western blot analysis, recognizing both isoforms of S6 kinase1 (p70S6K and p85S6K) and (ii) an ELISA based assay. The results of both assays were comparable. Since the two isoforms of S6 kinase1 are largely identical, the ELISA based method cannot differentiate between p706K and p85S6K. ELISA has the advantage that the amount of S6 kinase1 (p70S6K and p85S6K) can be determined and quantified. In this study, the average levels of phospho-p70 S6K were increased after PMA stimulation in Jurkat cells (unstimulated 4.18 vs. stimulated 7.65 U/100 mg protein) and in PBMCs (3.09 vs. 12.39 U/100 mg protein). Dose-dependent down-regulation of phospho-p70 S6K was found, induced by rapamycin in PMA-stimulated PBMCs (n = 20, P b 0.05) (Hartmann B, He X. et al., Abstract. Transplant International, Supp. s2 2010). In conclusion, the preliminary results of the studies mentioned above suggest that measuring the phosphorylation status of p70S6K could be helpful in monitoring the efficacy of therapy with mTOR inhibitors. For individualized immunosuppressive therapy it could provide information that is more relevant than sirolimus or everolimus trough concentrations. This pharmacodynamic approach could be advantageous for analyzing therapeutic efficacy in terms of tumor regression, at least in patients with Kaposi sarcoma, or for decreasing the chance of acute allograft rejection or drug side effects. Stable test systems, e.g. FACS analysis or possibly ELISA based tests, are needed to prove this approach prospectively in a larger, properly designed clinical trial. Abbreviations Akt protein kinase B AML acute myelogenous leukemia
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B. Hartmann / Clinica Chimica Acta 413 (2012) 1387–1390
CCI-779 ester of sirolimus cpm counts per min ELISA enzyme linked immunosorbent assay 4E-BP1 4E-binding protein 1 FACS fluorescence activated cell sorting FKBP FK binding protein Jurkat cells an immortalized line of T lymphocyte cells MDA-468breast cancer cell line mTOR mammalian target of rapamycin mTORC1 and 2 mTOR Complex 1 and 2 PBMC peripheral blood mononuclear cells PKCα protein kinase Cα PMA Phorbol 12-myristate 13-acetate p70S6K p70S6 Kinase p85S6K p85S6 Kinase p-S6RP phosphorylated S6 ribosomal protein RT room temperature S6K1 S6 Kinase 1 SCLC small cell lung cancer SGK1 serum- and glucocorticoid-induced protein kinase 1 T-cells T-lymphocytes Thr389 Threonine 389 = side of phosphorylation on the p70S6K
References [1] Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell 2006;124:471–84. [2] Loewith R, Jacinto E, Wullschleger S, et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell 2002;10:457–68. [3] Martel RR, Klicius J, Galet S. Inhibition of the immune response by rapamycin, a new antifungal antibiotic. Can J Physiol Pharmacol 1977;55:48–51. [4] Hara K, Maruki Y, Long X, et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 2002;110:177–89. [5] Kim DH, Sarbassov DD, Ali SM, et al. mTOR interacts with raptor to form a nutrientsensitive complex that signals to the cell growth machinery. Cell 2002;110:163–75. [6] Reinhard C, Fernandez A, Lamb NJ, Thomas G. Nuclear localization of p85s6k: functional requirement for entry into S phase. EMBO J 1994;13:1557–65. [7] Franco R, Rosenfeld MG. Hormonally inducible phosphorylation of a nuclear pool of ribosomal protein S6. J Biol Chem 1990;265:4321–5. [8] Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev 2004;18: 1926–45. [9] Morales JM, Wramner L, Kreis H, et al. Sirolimus does not exhibit nephrotoxicity compared to cyclosporine in renal transplant recipients. Am J Transplant 2002;2:436–42. [10] Pearce LR, Sommer EM, Sakamoto K, Wullschleger S, Alessi DR. Protor-1 is required for efficient mTORC2-mediated activation of SGK1 in the kidney. Biochem J 2011;436: 169–79. [11] Borders EB, Bivona C, Medina PJ. Mammalian target of rapamycin: biological function and target for novel anticancer agents. Am J Health Syst Pharm 2010;67: 2095–106.
[12] Campistol JM, Eris J, Oberbauer R, et al. Sirolimus therapy after early cyclosporine withdrawal reduces the risk for cancer in adult renal transplantation. J Am Soc Nephrol 2006;17:581–9. [13] Ruiz JC, Campistol JM, Sánchez-Fructuoso A, et al. Increase of proteinuria after conversion from calcineurin inhibitor to sirolimus-based treatment in kidney transplant patients with chronic allograft dysfunction. Nephrol Dial Transplant 2006;21:3252–7. [14] Ekberg H, Tedesco-Silva H, Demirbas A, Vítko S, Nashan B, Gürkan A. N Engl J Med 2007;357:2562–75. [15] Merkel S, Mogilevskaja N, Mengel M, Haller H, Schwarz A. Side effects of sirolimus. Transplant Proc 2006;38:714–5. [16] Dambrin C, Klupp J, Morris RE. Pharmacodynamics of immunosuppressive drugs. Curr Opin Immunol 2000;12:557–62. [17] Gallant HL, Yatscoff RW. P70 S6 kinase assay: a pharmacodynamic monitoring strategy for rapamycin; assay development. Transplant Proc 1996;28:3058–61. [18] Chung J, Kuo CJ, Crabtree GR, Blenis J. Rapamycin-FKBP specifically blocks growthdependent activation of and signaling by the 70 kd S6 protein kinases. Cell 1992;69: 1227–36. [19] Garcia-Paramio P, Cabrerizo Y, Bornancin F, Parker PJ. The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation. Biochem J 1998;333:631–6. [20] Pearson RB, Dennis PB, Han JW, et al. The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain. EMBO J 1995;14:5279–87. [21] Dennis PB, Pullen N, Kozma SC, Thomas G. The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases. Mol Cell Biol 1996;16:6242–51. [22] Seufferlein T, Rozengurt E. Rapamycin inhibits constitutive p70s6k phosphorylation, cell proliferation, and colony formation in small cell lung cancer cells. Cancer Res 1996;56:3895–7. [23] Peralba JM, DeGraffenried L, Friedrichs W, et al. Pharmacodynamic evaluation of CCI-779, an inhibitor of mTOR, in cancer patients. Clin Cancer Res 2003;9:2887–92. [24] Hartmann B, Schmid G, Graeb C, et al. Biochemical monitoring of mTOR inhibitorbased immunosuppression following kidney transplantation: a novel approach for tailored immunosuppressive therapy. Kidney Int 2005;68:2593–8. [25] Di Paolo S, Teutonico A, Ranieri E, Gesualdo L, Schena PF. Monitoring antitumor efficacy of rapamycin in Kaposi sarcoma. Am J Kidney Dis 2007;49:462–70. [26] Leogrande D, Teutonico A, Ranieri E, et al. Monitoring biological action of rapamycin in renal transplantation. Am J Kidney Dis 2007;50:314–25. [27] Dekter HE, Romijn FP, Temmink WP, van Pelt J, de Fijter JW, Smit NP. A spectrophotometric assay for routine measurement of mammalian target of rapamycin activity in cell lysates. Anal Biochem 2010;403:79–87. [28] Krutzik PO, Trejo A, Schulz KR, Nolan GP. Phospho flow cytometry methods for the analysis of kinase signaling in cell lines and primary human blood samples. Methods Mol Biol 2011;699:179–202. [29] Dieterlen M-T, Bittner HB, Klein S, Salisch S, Mittag A, Tárnok A, Dhein S, Mohr FW, Barten MJ. Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR-inhibitors in peripheral human blood. Cytometry Part B 2012;00B:000-000. [30] Perl AE, Kasner MT, Shank D, Luger SM, Carroll M. Single-cell pharmacodynamic monitoring of S6 ribosomal protein phosphorylation in AML blasts during a clinical trial combining the mTOR inhibitor sirolimus and intensive chemotherapy. Clin Cancer Res 2012;18:1716–25. [31] Weichart T, Säemann MD. The multiple facets of mTOR in immunity. Trends Immunol 2009;30:218–26.
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Invited critical review
p70S6 kinase phosphorylation for pharmacodynamic monitoring B. Hartmann ⁎ Zentrum für Innere Medizin, Klinik für Innere Medizin I, Sektion Nephrologie, Albert Einstein Allee 23, D-89070 Ulm, Germany
a r t i c l e
i n f o
Article history: Received 29 April 2011 Received in revised form 21 March 2012 Accepted 26 March 2012 Available online 13 April 2012 Keywords: mTOR inhibitor Rapamycin Sirolimus Everolimus Pharmacodynamic monitoring Kidney transplantation
a b s t r a c t Inhibitors of the mammalian target of rapamycin (mTOR) are administered as immunosuppressant as well as antineoplastic agents. Because of the narrow therapeutic index of mTOR inhibitors, drug monitoring is required, and this is usually done by measuring blood drug levels. Increasing knowledge of the signaling pathways of the mTOR protein kinase provides an opportunity for pharmacodynamic drug monitoring. With the different laboratory methods it is becoming possible to measure new biomarkers to control the influence of mTOR activity. One of these biomarkers is phospho-S6 kinase, with its isoform p70S6K. © 2012 Published by Elsevier B.V.
Contents
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1390
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase. It plays a central role in a great many cellular functions including metabolism, growth, survival, aging, synaptic plasticity and memory [1]. This signaling serine/threonine kinase is divided into two distinct kinase complexes, namely mTORC1 and mTORC2 [2]. The mTORC1 phosphorylates S6 kinase 1 with its isoforms p70S6 kinase (p70S6K) and p85S6K. The mTORC2 is rapamycin insensitive. It regulates phosphorylation of Akt, PKCα (protein kinase Cα) and SGK1 (serum- and glucocorticoid-induced protein kinase 1) and is thought to regulate actin cytoskeleton dynamics. mTOR inhibitors, namely sirolimus (rapamycin) and its derivative everolimus, target mTORC1. Rapamycin is a macrocyclic antibiotic, first described in 1975, produced by Streptomyces hygroscopicus. It was later renamed sirolimus because of its structural similarity to tacrolimus and because it was found to have immunosuppressive and antiproliferative properties [3]. Sirolimus and its derivative, everolimus, bind to the FK binding protein and inhibit mTORC1 mediated signal transduction pathways [2,4,5] (Fig. 1). mTORC1 controls the hydrophobic motif of p70 ribosomal S6
⁎ Tel.: + 49 731 500 44714; fax: + 49 731 500 44567. E-mail address: [email protected]. 0009-8981/$ – see front matter © 2012 Published by Elsevier B.V. doi:10.1016/j.cca.2012.03.023
kinase [4,5]. Two isoforms of S6K1 are produced from the same transcript by alternative initiation of translational start sites: the shorter form of S6K1, which is largely localized in the cytoplasm, is termed p70S6K. A second isoform, p85S6 kinase, is derived from the same gene and is identical to p70S6 kinase except for 23 extra residues at the amino terminus, which encode a nuclear localizing signal [6]. The functional significance of the differential subcellular localization of the two S6K1 isoforms has not been established, although it is tempting to speculate that the nuclear form is involved in phosphorylation of the nuclear pool of the free, chromatin-bound form of S6 [7]. p70S6K is probably one of the best characterized downstream effectors of mTORC1 [8]. In the late 1990s, clinical trials confirmed the efficacy of sirolimus as a potent immunosuppressant in renal transplantation without it appearing to cause a significant inherent nephrotoxicity [9]. Apart from immunosuppression in renal transplantation as an alternative to nephrotoxic calcineurin inhibitors, mTOR inhibitors are increasingly used in heart and liver transplantation. mTOR is also a key target for therapeutic intervention in cancer, as an important regulator of cell growth [10]. mTOR inhibitors are used, for example, for chemotherapy of soft tissue and bone sarcomas [11]. Sirolimus and everolimus are critical dose drugs with a low therapeutic index. Under or overdosing can lead respectively to rejection or toxicity. mTOR inhibitors are associated with a number of possible
1388
B. Hartmann / Clinica Chimica Acta 413 (2012) 1387–1390
A simplyfied model of the mTOR pathway Cellular stress, Cellular nutrition, Insulin, ILGF, etc.
Sirolimus or Everolimus
+ Sirolimus or Everolimus
-
+
mTORC1
FKBP
-
mTORC2
FKBP
P
P
4E-BP1
Protein synthesis
p70S6K
Cell growth
Cell proliferation
Fig. 1. A simplified model of the mTOR pathway: mTOR has a central role in cell metabolism and is found in two distinct protein complexes (mTORC1 and mTORC2). Only mTORC1 is sensitive to rapamycin (i.e. sirolimus and everolimus). Active mTORC1 phosphorylates its downstream substrates 4E-BP1 and p70S6K, thus stimulating protein synthesis and cell growth. mTOR inhibitors such as sirolimus and everolimus bind to the FK binding protein 12 (FKBP12) and, as this complex, inhibit mTORC1 and its downstream effects. Adapted from [31].
adverse effects, including leukopenia, thrombocytopenia, anemia, hypercholesterolemia, hypertriglyceridemia and diarrhea, as well as wound healing problems, interstitial pneumonitis and proteinuria. Although there are many benefits of using mTOR inhibitors in renal transplantation, approximately 30–50% of patients on mTOR inhibitor therapy discontinue it during follow-up due to the related side effects [12–15]. Adjustment of mTOR inhibitor doses currently relies entirely on sirolimus or everolimus blood concentrations, which may not necessarily correlate with the pharmacological effects of the drugs on immune cells. Recommended trough levels for sirolimus, derived from a ten-year perspective, are at present from 5 to 15 ng/ml, which is too wide a range for reliable immunosuppressive therapy. Knowledge of the pharmacodynamic processes of immunosuppressive agents offers the opportunity of estimating the clinical effect and thereby of further refining and optimizing current therapy [16]. The phosphorylation of p70S6K as a well characterized downstream effect of mTOR makes measuring the activity of this enzyme a suitable target for pharmacodynamic monitoring of mTOR inhibition [17,18]. Studies of rapamycin-sensitive regulatory phosphorylation sites showed that rapamycin-sensitive sites were phosphorylated in response to mitogenic stimulation [19] and that the three sites underwent hierarchical dephosphorylation by rapamycin treatment, with T389 > S404 > T229, T389 dephosphorylation most closely paralleling loss of kinase activity [20]. Conversion of these sites to either acidic or neutral amino acids revealed that T229 and T389 were critical regulatory sites, whereas S404 appeared to play a modulatory role. Mutational analysis also revealed that the principal site of rapamycin inducing S6K1 inactivation was T389, and substitution of an acidic residue in this position resulted in a kinase variant which increased basal activity and was largely rapamycin resistant [21]. Different approaches have already been attempted to establish the usefulness of pharmacodynamic monitoring of mTOR inhibition on the basis of measuring p70S6K activity. This was estimated in white blood cells after treatment with rapamycin using a radioactivelabeled phospho-p70S6K antiserum. Although assay robustness was poor, it was possible to show a difference between lower and higher mTOR inhibition, as well as lack of influence on cells treated with calcineurin inhibitors in vitro by measuring the counts per min (cpm) [17].
The role of p70S6K activity in promoting the growth of small cell lung cancer (SCLC) was evaluated in cell growth experiments, in colony formation assays and by measuring p70S6K activity with Western blot analysis after treatment of SCLC cell lines with different concentrations of rapamycin. Cell growth, colony forming factor and phosphorylation status of p70S6K were reduced with increasing sirolimus concentrations [22]. In 2003, Peralba et al. [23] reported that the treatment with CCI-779, an ester of sirolimus, of nude mice bearing the CCI-779 susceptible breast cancer cell line MDA-468 resulted in high inhibition of p70S6K activity in peripheral blood mononuclear cells (PBMC). Furthermore, the degree of inhibition was identical in PBMC and simultaneously collected tumor tissue, suggesting that the PBMCs were an adequate surrogate tissue for p70S6K activity in vivo. The activity of the p70S6K was measured by Western blot analysis. Sirolimus based immunosuppressive therapy in renal transplant patients was monitored in a small study for the first time by measuring the trough level of sirolimus and by assessing the phosphorylation status at the Thr389 site of p70S6K in PBMCs using Western blot [24]. A total of 36 patients with renal transplants and 8 healthy controls were enrolled. Results suggested that sirolimus treatment was associated with pronounced inhibition of p70S6K phosphorylation, compared with results from healthy controls or patients treated with other immunosuppressant therapy. Remarkably, no correlation of the sirolimus trough level with reduction in phospho-p70S6K could be demonstrated. Furthermore, some patients exhibited low trough concentrations yet a strong reduction in phospho-p70S6K, indicating individual enzyme susceptibility. There were five episodes of clinically relevant rejection during the study period. All the patients who experienced acute rejection uniformly maintained a high degree of enzyme phosphorylation, whereas non-rejecters showed significant inhibition of phosphorylation. A reduction in p70S6K phosphorylation status below 60% in comparison with the results from healthy controls was considered as significant in that survey. In all patients with a trough level equal or above 6 ng/mL, p70S6K phosphorylation was constantly inhibited. This study concluded that the phosphorylation status of p70S6K appeared to provide more relevant information on the desired effect of sirolimus in target cells than trough concentration measurements.
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Apart from their capacity for immunosuppression, mTOR inhibitors are being increasingly recognized as an anticancer therapy. Cancer is also a side effect of long-term immunosuppression. The prevalence of some cancers, especially skin cancer, is increased in transplanted patients, so it is important to know which cancer will be sensitive to rapamycin treatment. The pharmacodynamic approach implies the need for biochemical monitoring of immunosuppressive therapy based on mTOR inhibitors, as well as identification of increased mTOR activity due to growth of the cancer. In a study with renal transplant recipients with Kaposi sarcoma, markedly increased mTOR activity was shown by measuring increased p70S6K activity by Western blot analysis [25]. p70S6K phosphorylation was estimated in PBMCs from 37 patients being treated with a cyclosporine-based immunosuppressive therapy, of whom ten had Kaposi sarcoma. Six months after switching to rapamycin therapy, p70S6K activity was substantially reduced in both groups. This reduction was associated with regression of the dermal neoplasm in the patients with Kaposi sarcoma. Over-activation of basal p70S6K in PBMCs from renal transplant recipients thus appeared to be associated with Kaposi sarcoma dermal lesions, and inhibition of p70S6K was linked to regression of skin cancer lesions. The influence of co-medication with tacrolimus on the potency of sirolimus for reducing phosphorylation of p70S6K was investigated in another study with kidney transplant patients [26]. The activity of p70S6K in PBMCs was also measured by Western blotting and the results were compared with the sirolimus trough level measured in parallel. From this survey it could be concluded that co-medication with tacrolimus limited the inhibitory effect of sirolimus on p70S6K activation and – as had been shown by others before – there is no correlation between p70S6K phosphorylation and trough levels, because of high individual variation in p70S6K phosphorylation. In all the studies mentioned above, Western blot analysis was used to measure the activity of p70S6K. PBMCs were isolated from a freshly drawn blood by means of Ficoll-Hypaque gradient centrifugation. The procedure was performed in the presence of protease and phosphatase inhibitor cocktails. The cells were then lysed and subjected to blotting as described in the literature. Monoclonal antiphospho-p70S6K antibodies were applied directed against the phosphorylation sites serine 411 or threonine 389. In some studies, p70S6K generation was stimulated in vivo as well as in vitro by growth factors such as insulin. The different procedures to analyze the time from drawing to lysing the blood were not described. Nor was there any information about inter- and intraassay variation. The Western blot technique has the advantage of not being very expensive and it is an established test in any laboratory. Protein immunoblot analysis is however not a quantitative technique and is not a suitable test for large population studies and inter-subject comparisons. As an alternative to p70S6K measurement there is a possibility to measure the mTOR activity itself. There is an ELISA-based assay for measuring the kinase activity of mTOR. This test can be applied for in vitro mTOR inhibitor screening and for assessing the regulation of mTOR cell signaling. With this method mTOR activity was studied in kidney transplant recipients in one study [27]. PBMC samples from five renal allograft patients were obtained before treatment with everolimus and one month after the start of everolimus therapy. With this approach, no significant difference in mTOR activity could be seen between the two groups. The conclusion from these preliminary results is that there is no advantage in directly measuring mTOR activity for measuring p70S6K activity in order to monitor the biochemical effect of mTOR inhibitor therapy. Two other assays in particular, FACS analysis and ELISA, are presently being studied for analyzing p70S6K activity. The results of phosphorylated S6 ribosomal protein (p-S6RP) measurement by FACS analysis have recently been published. Phospho-specific flow cytometry, or phospho flow, measures the phosphorylation status of intracellular proteins at the single cell
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level [28]. In one study to evaluate the eligibility of this analytical procedure, blood from five volunteers was analyzed. The blood was incubated with different clinically relevant concentrations of either sirolimus or other immunosuppressive drugs, e.g. cyclosporine A, mycophenolic acid or dexamethasone. Following drug treatment, the whole blood samples were stimulated by adding phorbol 12-myristate 13-acetate (PMA) and ionomycin, in order to increase the amount of p-S6RP. After this procedure the whole blood was analyzed by flow cytometry to measure p-S6RP in T-cells. As expected, treatment with sirolimus showed suppression of p-S6RP. Neither cyclosporine A, mycophenolic acid nor dexamethasone had any effect on mTOR-related p-S6RP. In this small survey with blood samples from five volunteers, intra and interassay, as well as interindividual variability were examined. The results were 3 to 5%, 12 to 25% and 14 to 38%, respectively. Furthermore, the time from blood collection to analyzing it was measured under different conditions (room temperature, 4 °C). It could been shown that samples can be stored at RT or 4 °C for up to 2 h after withdrawal [29]. Blood samples from patients with acute myelogenous leukemia (AML) were analyzed with the same technique to measure p-S6RP. The mTOR signaling pathway is frequently activated in AML, but it has been stated that S6 phosphorylation in AML blasts is heterogeneous and, in some cases, intrinsically resistant to rapamycin at clinical concentrations. The mTOR inhibitor sirolimus was added to chemotherapy with mitoxantrone, etoposide and cytarabine. Inhibition of p-S6RP in the majority of subjects' tumor cells could be shown during therapy using a whole blood fixation/permeabilization technique for flow cytometry [30]. According to the results of the two studies, it seems that phospho-specific flow cytometry is a reliable test for measuring p-S6RP in T-cells. Its advantage is that the target cells (T-lymphocytes) can be measured directly, since the test allows the phosphorylation status of intracellular proteins to be measured at the single cell level. ELISA based measurement of p70SK is also possible. In a cell culture model (Jurkat cells and PBMCs from buffy coats), the phosphorylation status was analyzed of p70S6K. Following treatment of the cells with sirolimus, they were stimulated with the phorbol ester PMA. Phospho-p70S6K expression was measured by (i) semiquantitative Western blot analysis, recognizing both isoforms of S6 kinase1 (p70S6K and p85S6K) and (ii) an ELISA based assay. The results of both assays were comparable. Since the two isoforms of S6 kinase1 are largely identical, the ELISA based method cannot differentiate between p706K and p85S6K. ELISA has the advantage that the amount of S6 kinase1 (p70S6K and p85S6K) can be determined and quantified. In this study, the average levels of phospho-p70 S6K were increased after PMA stimulation in Jurkat cells (unstimulated 4.18 vs. stimulated 7.65 U/100 mg protein) and in PBMCs (3.09 vs. 12.39 U/100 mg protein). Dose-dependent down-regulation of phospho-p70 S6K was found, induced by rapamycin in PMA-stimulated PBMCs (n = 20, P b 0.05) (Hartmann B, He X. et al., Abstract. Transplant International, Supp. s2 2010). In conclusion, the preliminary results of the studies mentioned above suggest that measuring the phosphorylation status of p70S6K could be helpful in monitoring the efficacy of therapy with mTOR inhibitors. For individualized immunosuppressive therapy it could provide information that is more relevant than sirolimus or everolimus trough concentrations. This pharmacodynamic approach could be advantageous for analyzing therapeutic efficacy in terms of tumor regression, at least in patients with Kaposi sarcoma, or for decreasing the chance of acute allograft rejection or drug side effects. Stable test systems, e.g. FACS analysis or possibly ELISA based tests, are needed to prove this approach prospectively in a larger, properly designed clinical trial. Abbreviations Akt protein kinase B AML acute myelogenous leukemia
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CCI-779 ester of sirolimus cpm counts per min ELISA enzyme linked immunosorbent assay 4E-BP1 4E-binding protein 1 FACS fluorescence activated cell sorting FKBP FK binding protein Jurkat cells an immortalized line of T lymphocyte cells MDA-468breast cancer cell line mTOR mammalian target of rapamycin mTORC1 and 2 mTOR Complex 1 and 2 PBMC peripheral blood mononuclear cells PKCα protein kinase Cα PMA Phorbol 12-myristate 13-acetate p70S6K p70S6 Kinase p85S6K p85S6 Kinase p-S6RP phosphorylated S6 ribosomal protein RT room temperature S6K1 S6 Kinase 1 SCLC small cell lung cancer SGK1 serum- and glucocorticoid-induced protein kinase 1 T-cells T-lymphocytes Thr389 Threonine 389 = side of phosphorylation on the p70S6K
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