essential role for the p110 isoform in phosphoinositide 3-kinase activation and cell proliferation in acute myeloid leukemia
http://www.100md.com
《血液学杂志》
the Departement d'Hematologie, INSeRM U567, CNRS UMR 8104, Universite Paris Descartes 5, Institut Cochin
Service d'Hematologie and the Departement de Statistique, hospital Cochin, AP-HP, Paris, France
Laboratoire d'Hematologie, hospital de Reims, Reims, France
ICOS Corporation, Bothell, Washington
Ludwig Institute for Cancer Research and the Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom
Groupe Ouest-est des Leucemies et des Autres Maladies du Sang (GOeLAMS), Tours, France.
Abstract
The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been shown to be frequently activated in blast cells from patients with acute myeloid leukemia (AML) and to contribute to survival and proliferation of these cells. Of the 8 distinct mammalian isoforms of PI3K, it is the class I PI3Ks (p110, p110, p110, and p110) that are responsible for Akt activation. It is not known which PI3K isoform is critical in AML. Here we show that the p110 isoform of PI3K is consistently expressed at a high level in blast cells from AML, in contrast to the other class I isoforms, the expression of which was very variable among patients. IC87114, a p110-selective inhibitor, suppressed both constitutive and Flt-3–stimulated Akt activation in blasts to the same extent as Ly294002, an inhibitor of all PI3K isoforms. Moreover, IC87114 inhibited AML cell proliferation without affecting the proliferation of normal hematopoietic progenitor cells. These observations identify p110 as a potential therapeutic target in AML.
Introduction
AML is a clonal hematologic disease and is due to acquired mutations in immature progenitors, resulting in a block of differentiation leading to an accumulation of myeloid blasts.1 Two classes of mutations, one impairing cell differentiation and the other conferring survival and proliferative benefits, are known to cooperate to cause acute leukemia.2 Phosphoinositide 3-kinase (PI3K) and its downstream target Akt have been reported to be frequently constitutively activated in leukemic blasts from patients with AML and to contribute to cell survival and proliferation.3-5 Moreover, constitutive phosphorylation of Akt or of forkhead in rabdomyosarcoma (FKHR), one of its substrates, correlates with decreased survival in patients with AML.3,6 Among the 8 isoforms of PI3K, the class I PI3Ks are responsible for Akt activation in cells. These PI3Ks are heterodimers composed of a catalytic and a regulatory subunit. The class IA PI3K catalytic subunits (p110, p110, and p110) associate with Src homology domain 2 (SH2)–containing regulatory subunits and signal downstream of cytokine and tyrosine kinase receptors. p110 is the only class IB PI3K, and functions in the context of heterotrimeric G-protein–coupled receptor signaling. p110 and - are widely distributed in mammalian tissues, whereas p110 and - show a more restricted distribution and are mainly but not exclusively expressed in blood cells and their precursors.7 In this study, we examined which of the class I PI3K isoforms is responsible for PI3K activation in AML blasts.
Study design
Patients
Cells
Bone marrow cells from newly diagnosed patients with AML were obtained before induction of chemotherapy. Bone marrow samples were subjected to Ficoll-Hypaque density gradient separation to isolate mononuclear cells (BMMCs). CD34+ cells from cord blood were isolated as previously reported.9 The OPM2 cell line was established from the peripheral blood of a patient with multiple myeloma.10
Reagents
The p110 inhibitor IC87114 was from ICOS Corporation, Bothell, WA.11
Western blot analysis
BMMCs of patients with AML were starved in serum-free medium for 4 hours. Cells were incubated with or without inhibitor for 30 minutes at 37°C. Cells were then boiled in Laemmli sample buffer and proteins were analyzed by Western blot. enhanced chemiluminescence (eCL; Amersham Pharmacia Biotech, Piscataway, NJ) or SuperSignal West Femto (Pierce, Rockford, IL) chemoluminescence kits were used for detection. Western blots were quantified using the ImageJ 1.32 software (National Institutes of Health, Bethesda, MD) after densitometric scanning of the films.
Cell proliferation assays
BMMCs were cultured in -medium with 5% fetal calf serum (FCS) with or without FLT-3 ligand (10 ng/mL) for 48 hours and with or without 10 μM IC87114. [3H]-thymidine (1 μCi [37 kBq]) was added for a final 6 hours and the amount of radioactivity incorporated was determined by trichloracetic acid precipitation. CD34+ cells from cord blood were cultured in stem cell factor (SCF; 20 ng/mL), FLT-3 ligand (10 ng/mL), and Tpo (20 nM) for 48 hours with or without 10 μM IC87114 and pulsed for 12 hours with [3H]-thymidine.
Results and discussion
p110 is the only class I PI3K isoform consistently present in AML blasts
Inhibition of Akt phosphorylation by IC87114, a p110-selective inhibitor
Akt phosphorylation in AML blasts increases in response to FLT-3 ligand.12 We tested the impact of p110 inhibition on Akt phosphorylation. Figure 2B shows that FLT-3 ligand–stimulated Akt phosphorylation was inhibited by IC87114 to the same extent as by LY294002, showing that p110 can be responsible for PI3K activation after FLT-3 ligand stimulation. At present, it is not clear why p110 is also the main contributor of PI3K activity in cells that also express p110 and p110. One possible explanation would be that the expression level of p110 is significantly higher than that of the other isoforms.
Inhibition of AML cell proliferation by IC87114
Next, we tested the effect of IC87114 on cell proliferation on the blast samples described in the previous paragraph. AML proliferation was found to be almost completely blocked by 10 μM IC87114. IC87114 also strongly reduced the proliferation of cells stimulated with FLT-3 ligand (Figure 2D). We observed that FLT-3 ligand was still able to stimulate both PI3K activity and proliferation of blast cells from patient no. 102, who presented with an activating mutation (ITD) of FLT-3 (Figure 2B,D), confirming the observations previously reported by Bruserud et al.13 In contrast, cell proliferation of OMP2 and cord blood CD34+ cells was not decreased by IC87114 (data not shown).
AML is associated with poor long-term survival. The development of new therapeutic strategies directed against specific targets is an area of intense interest and may prove effective as adjunct treatments in combination with traditional chemotherapy. The PI3K/Akt pathway is often activated in AML blast cells, contributing to their survival4,5 and their proliferation (results described in this manuscript). Blockade of all PI3K isoforms in the organism using nonselective PI3K inhibitors such as LY294002 or wortmannin is very toxic in vivo, possibly due to a general requirement of PI3K for many cellular functions. Mice lacking functional p110 are viable and fertile, in contrast to mice lacking p110 or p110, which are embryonic lethal.14-17 This finding suggests that specifically blocking p110 might be less toxic than inhibiting all PI3K activities. Our data suggest that in patients with AML, pharmacologic inhibition of p110 may offer clinical benefit.
Acknowledgements
We thank Dr R. Wetzker (University of Jena, Germany) for providing the anti-p110 antibody.
Footnotes
Prepublished online as Blood First edition Paper, April 19, 2005; DOI 10.1182/blood-2004-08-3225.
Supported by the Comite de Paris of the Ligue Nationale Contre le Cancer (LNCC; laboratoire associe no. 8), the Association pour la Recherche contre le Cancer (ARC) and the Groupe Ouest-est des Leucemies et des Autres Maladies du Sang (GOeLAMS).
P.S. and V.B. contributed equally to this work.
One of the authors, J.S.H., is employed by the ICOS Corporation company, whose product (IC87114 inhibitor) is used in the present work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
References
Passegue e, Jamieson CH, Ailles Le, Weissman IL. Normal and leukemic hematopoiesis: are leukemias a stem cell disorder or a reacquisition of stem cell characteristics? Proc Natl Acad Sci U S A. 2003;100(suppl 1): 11842-11849.
Gilliland DG, Tallman MS. Focus on acute leukemias. Cancer Cell. 2002;1: 417-420.
Min YH, eom JI, Cheong JW, et al. Constitutive phosphorylation of Akt/PKB protein in acute myeloid leukemia: its significance as a prognostic variable. Leukemia. 2003;17: 995-997.
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.
Zhao S, Konopleva M, Cabreira-Hansen M, et al. Inhibition of phosphatidylinositol 3-kinase dephosphorylates BAD and promotes apoptosis in myeloid leukemias. Leukemia. 2004;18: 267-275.
Cheong JW, eom JI, Maeng HY, et al. Constitutive phosphorylation of FKHR transcription factor as a prognostic variable in acute myeloid leukemia. Leuk Res. 2003;27: 1159-1162.
Sawyer C, Sturge J, Bennett DC, et al. Regulation of breast cancer cell chemotaxis by the phosphoinositide 3-kinase p110delta. Cancer Res. 2003;63: 1667-1675.
Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classification of acute myeloid leukemia: a report of the French-American-British Cooperative Group. Ann Intern Med. 1985;103: 620-625.
Freyssinier JM, Lecoq-Lafon C, Amsellem S, et al. Purification, amplification and characterization of a population of human erythroid progenitors. Br J Haematol. 1999;106: 912-922.
Katagiri S, Yonezawa T, Kuyama J, et al. Two distinct human myeloma cell lines originating from one patient with myeloma. Int J Cancer. 1985;36: 241-246.
Sadhu C, Dick K, Tino WT, Staunton De. Selective role of PI3K delta in neutrophil inflammatory responses. Biochem Biophys Res Commun. 2003;308: 764-769.
Meyer C, Drexler HG. FLT3 ligand inhibits apoptosis and promotes survival of myeloid leukemia cell lines. Leuk Lymphoma. 1999;32: 577-581.
Bruserud O, Hovland R, Wergeland L, Huang TS, Gjertsen BT. Flt3-mediated signaling in human acute myelogenous leukemia (AML) blasts: a functional characterization of Flt3-ligand effects in AML cell populations with and without genetic Flt3 abnormalities. Haematologica. 2003;88: 416-428.
Ali K, Bilancio A, Thomas M, et al. essential role for the p110delta phosphoinositide 3-kinase in the allergic response. Nature. 2004;431: 1007-1011.
Clayton e, Bardi G, Bell Se, et al. A crucial role for the p110delta subunit of phosphatidylinositol 3-kinase in B cell development and activation. J exp Med. 2002;196: 753-763.
Jou ST, Carpino N, Takahashi Y, et al. essential, nonredundant role for the phosphoinositide 3-kinase p110delta in signaling by the B-cell receptor complex. Mol Cell Biol. 2002;22: 8580-8591.
Okkenhaug K, Bilancio A, Farjot G, et al. Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice. Science. 2002;297: 1031-1034.
Vanhaesebroeck B, Welham MJ, Kotani K, et al. P110delta, a novel phosphoinositide 3-kinase in leukocytes. Proc Natl Acad Sci U S A. 1997;94: 4330-4335.
Pietrucha R, Rubio I, Wymann MP, Wetzker R. Phosphoinositide 3-kinase gamma mediates Jun kinase activation via its lipid-kinase activity. Adv enzyme Regul. 2004;44: 299-308.(Pierre Sujobert, Valerie )
Service d'Hematologie and the Departement de Statistique, hospital Cochin, AP-HP, Paris, France
Laboratoire d'Hematologie, hospital de Reims, Reims, France
ICOS Corporation, Bothell, Washington
Ludwig Institute for Cancer Research and the Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom
Groupe Ouest-est des Leucemies et des Autres Maladies du Sang (GOeLAMS), Tours, France.
Abstract
The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been shown to be frequently activated in blast cells from patients with acute myeloid leukemia (AML) and to contribute to survival and proliferation of these cells. Of the 8 distinct mammalian isoforms of PI3K, it is the class I PI3Ks (p110, p110, p110, and p110) that are responsible for Akt activation. It is not known which PI3K isoform is critical in AML. Here we show that the p110 isoform of PI3K is consistently expressed at a high level in blast cells from AML, in contrast to the other class I isoforms, the expression of which was very variable among patients. IC87114, a p110-selective inhibitor, suppressed both constitutive and Flt-3–stimulated Akt activation in blasts to the same extent as Ly294002, an inhibitor of all PI3K isoforms. Moreover, IC87114 inhibited AML cell proliferation without affecting the proliferation of normal hematopoietic progenitor cells. These observations identify p110 as a potential therapeutic target in AML.
Introduction
AML is a clonal hematologic disease and is due to acquired mutations in immature progenitors, resulting in a block of differentiation leading to an accumulation of myeloid blasts.1 Two classes of mutations, one impairing cell differentiation and the other conferring survival and proliferative benefits, are known to cooperate to cause acute leukemia.2 Phosphoinositide 3-kinase (PI3K) and its downstream target Akt have been reported to be frequently constitutively activated in leukemic blasts from patients with AML and to contribute to cell survival and proliferation.3-5 Moreover, constitutive phosphorylation of Akt or of forkhead in rabdomyosarcoma (FKHR), one of its substrates, correlates with decreased survival in patients with AML.3,6 Among the 8 isoforms of PI3K, the class I PI3Ks are responsible for Akt activation in cells. These PI3Ks are heterodimers composed of a catalytic and a regulatory subunit. The class IA PI3K catalytic subunits (p110, p110, and p110) associate with Src homology domain 2 (SH2)–containing regulatory subunits and signal downstream of cytokine and tyrosine kinase receptors. p110 is the only class IB PI3K, and functions in the context of heterotrimeric G-protein–coupled receptor signaling. p110 and - are widely distributed in mammalian tissues, whereas p110 and - show a more restricted distribution and are mainly but not exclusively expressed in blood cells and their precursors.7 In this study, we examined which of the class I PI3K isoforms is responsible for PI3K activation in AML blasts.
Study design
Patients
Cells
Bone marrow cells from newly diagnosed patients with AML were obtained before induction of chemotherapy. Bone marrow samples were subjected to Ficoll-Hypaque density gradient separation to isolate mononuclear cells (BMMCs). CD34+ cells from cord blood were isolated as previously reported.9 The OPM2 cell line was established from the peripheral blood of a patient with multiple myeloma.10
Reagents
The p110 inhibitor IC87114 was from ICOS Corporation, Bothell, WA.11
Western blot analysis
BMMCs of patients with AML were starved in serum-free medium for 4 hours. Cells were incubated with or without inhibitor for 30 minutes at 37°C. Cells were then boiled in Laemmli sample buffer and proteins were analyzed by Western blot. enhanced chemiluminescence (eCL; Amersham Pharmacia Biotech, Piscataway, NJ) or SuperSignal West Femto (Pierce, Rockford, IL) chemoluminescence kits were used for detection. Western blots were quantified using the ImageJ 1.32 software (National Institutes of Health, Bethesda, MD) after densitometric scanning of the films.
Cell proliferation assays
BMMCs were cultured in -medium with 5% fetal calf serum (FCS) with or without FLT-3 ligand (10 ng/mL) for 48 hours and with or without 10 μM IC87114. [3H]-thymidine (1 μCi [37 kBq]) was added for a final 6 hours and the amount of radioactivity incorporated was determined by trichloracetic acid precipitation. CD34+ cells from cord blood were cultured in stem cell factor (SCF; 20 ng/mL), FLT-3 ligand (10 ng/mL), and Tpo (20 nM) for 48 hours with or without 10 μM IC87114 and pulsed for 12 hours with [3H]-thymidine.
Results and discussion
p110 is the only class I PI3K isoform consistently present in AML blasts
Inhibition of Akt phosphorylation by IC87114, a p110-selective inhibitor
Akt phosphorylation in AML blasts increases in response to FLT-3 ligand.12 We tested the impact of p110 inhibition on Akt phosphorylation. Figure 2B shows that FLT-3 ligand–stimulated Akt phosphorylation was inhibited by IC87114 to the same extent as by LY294002, showing that p110 can be responsible for PI3K activation after FLT-3 ligand stimulation. At present, it is not clear why p110 is also the main contributor of PI3K activity in cells that also express p110 and p110. One possible explanation would be that the expression level of p110 is significantly higher than that of the other isoforms.
Inhibition of AML cell proliferation by IC87114
Next, we tested the effect of IC87114 on cell proliferation on the blast samples described in the previous paragraph. AML proliferation was found to be almost completely blocked by 10 μM IC87114. IC87114 also strongly reduced the proliferation of cells stimulated with FLT-3 ligand (Figure 2D). We observed that FLT-3 ligand was still able to stimulate both PI3K activity and proliferation of blast cells from patient no. 102, who presented with an activating mutation (ITD) of FLT-3 (Figure 2B,D), confirming the observations previously reported by Bruserud et al.13 In contrast, cell proliferation of OMP2 and cord blood CD34+ cells was not decreased by IC87114 (data not shown).
AML is associated with poor long-term survival. The development of new therapeutic strategies directed against specific targets is an area of intense interest and may prove effective as adjunct treatments in combination with traditional chemotherapy. The PI3K/Akt pathway is often activated in AML blast cells, contributing to their survival4,5 and their proliferation (results described in this manuscript). Blockade of all PI3K isoforms in the organism using nonselective PI3K inhibitors such as LY294002 or wortmannin is very toxic in vivo, possibly due to a general requirement of PI3K for many cellular functions. Mice lacking functional p110 are viable and fertile, in contrast to mice lacking p110 or p110, which are embryonic lethal.14-17 This finding suggests that specifically blocking p110 might be less toxic than inhibiting all PI3K activities. Our data suggest that in patients with AML, pharmacologic inhibition of p110 may offer clinical benefit.
Acknowledgements
We thank Dr R. Wetzker (University of Jena, Germany) for providing the anti-p110 antibody.
Footnotes
Prepublished online as Blood First edition Paper, April 19, 2005; DOI 10.1182/blood-2004-08-3225.
Supported by the Comite de Paris of the Ligue Nationale Contre le Cancer (LNCC; laboratoire associe no. 8), the Association pour la Recherche contre le Cancer (ARC) and the Groupe Ouest-est des Leucemies et des Autres Maladies du Sang (GOeLAMS).
P.S. and V.B. contributed equally to this work.
One of the authors, J.S.H., is employed by the ICOS Corporation company, whose product (IC87114 inhibitor) is used in the present work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
References
Passegue e, Jamieson CH, Ailles Le, Weissman IL. Normal and leukemic hematopoiesis: are leukemias a stem cell disorder or a reacquisition of stem cell characteristics? Proc Natl Acad Sci U S A. 2003;100(suppl 1): 11842-11849.
Gilliland DG, Tallman MS. Focus on acute leukemias. Cancer Cell. 2002;1: 417-420.
Min YH, eom JI, Cheong JW, et al. Constitutive phosphorylation of Akt/PKB protein in acute myeloid leukemia: its significance as a prognostic variable. Leukemia. 2003;17: 995-997.
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.
Zhao S, Konopleva M, Cabreira-Hansen M, et al. Inhibition of phosphatidylinositol 3-kinase dephosphorylates BAD and promotes apoptosis in myeloid leukemias. Leukemia. 2004;18: 267-275.
Cheong JW, eom JI, Maeng HY, et al. Constitutive phosphorylation of FKHR transcription factor as a prognostic variable in acute myeloid leukemia. Leuk Res. 2003;27: 1159-1162.
Sawyer C, Sturge J, Bennett DC, et al. Regulation of breast cancer cell chemotaxis by the phosphoinositide 3-kinase p110delta. Cancer Res. 2003;63: 1667-1675.
Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classification of acute myeloid leukemia: a report of the French-American-British Cooperative Group. Ann Intern Med. 1985;103: 620-625.
Freyssinier JM, Lecoq-Lafon C, Amsellem S, et al. Purification, amplification and characterization of a population of human erythroid progenitors. Br J Haematol. 1999;106: 912-922.
Katagiri S, Yonezawa T, Kuyama J, et al. Two distinct human myeloma cell lines originating from one patient with myeloma. Int J Cancer. 1985;36: 241-246.
Sadhu C, Dick K, Tino WT, Staunton De. Selective role of PI3K delta in neutrophil inflammatory responses. Biochem Biophys Res Commun. 2003;308: 764-769.
Meyer C, Drexler HG. FLT3 ligand inhibits apoptosis and promotes survival of myeloid leukemia cell lines. Leuk Lymphoma. 1999;32: 577-581.
Bruserud O, Hovland R, Wergeland L, Huang TS, Gjertsen BT. Flt3-mediated signaling in human acute myelogenous leukemia (AML) blasts: a functional characterization of Flt3-ligand effects in AML cell populations with and without genetic Flt3 abnormalities. Haematologica. 2003;88: 416-428.
Ali K, Bilancio A, Thomas M, et al. essential role for the p110delta phosphoinositide 3-kinase in the allergic response. Nature. 2004;431: 1007-1011.
Clayton e, Bardi G, Bell Se, et al. A crucial role for the p110delta subunit of phosphatidylinositol 3-kinase in B cell development and activation. J exp Med. 2002;196: 753-763.
Jou ST, Carpino N, Takahashi Y, et al. essential, nonredundant role for the phosphoinositide 3-kinase p110delta in signaling by the B-cell receptor complex. Mol Cell Biol. 2002;22: 8580-8591.
Okkenhaug K, Bilancio A, Farjot G, et al. Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice. Science. 2002;297: 1031-1034.
Vanhaesebroeck B, Welham MJ, Kotani K, et al. P110delta, a novel phosphoinositide 3-kinase in leukocytes. Proc Natl Acad Sci U S A. 1997;94: 4330-4335.
Pietrucha R, Rubio I, Wymann MP, Wetzker R. Phosphoinositide 3-kinase gamma mediates Jun kinase activation via its lipid-kinase activity. Adv enzyme Regul. 2004;44: 299-308.(Pierre Sujobert, Valerie )