Letters to the Editor
405
Acetylation of FOXO3a transcription factor in response to imatinib of chronic myeloid leukemia
Leukemia (2009) 23, 405–406; doi:10.1038/leu.2008.186; published online 7 August 2008
Members of the FOXO subfamily of forkhead transcription factors, including FOXO1 (FKHR), FOXO3a (FKHRL1), FOXO4 (AFX) and FOXO6 in humans, are key regulators of cell cycle progression, DNA damage repair, reactive oxygen species detoxification, apoptosis and autophagy. Post-translational modifications (phosphorylation, acetylation and ubiquitination) regulate the transcriptional activity of FOXO proteins by directing their subcellular location, DNA binding and stability.1 Phosphorylation by the phosphatidyl-inositol 3-OH kinase/AKT axis at three highly conserved AKT recognition sites within N- and C-terminal regions is the most extensively studied posttranslational modification of FOXO proteins. Two phosphorylation sites (Thr24 and Ser256 in FOXO1 corresponding to Thr32 and Ser253 in FOXO3a) create binding motifs for the 14-3-3 scaffolding proteins thereby causing the translocation of FOXO and its retention in the cytoplasm.1 Moreover, 14-3-3 binding to phosphorylated Ser253 in FOXO3a masks a cluster of three adjacent basic residues (Arg248, Arg249 and Arg250), thereby weakening its DNA-binding affinity.2 The third phosphorylation site (Ser319 in FOXO1 corresponding to Ser315 in FOXO3a) creates a consensus sequence for casein kinase 1 promoting further phosphorylations at serine residues that potentiate the nuclear export of FOXO through interactions with the export machinery.1 More recently, acetylation has been involved in the subcellular location and transcriptional regulation of FOXO. It is induced in response to oxidative stress by enhanced interactions with histone acetyltransferases p300/c-AMPresponse element-binding protein-binding protein (CBP) and p300/CBP-associated factor and promotes the nuclear import of FOXO.3 Following acetylation, FOXO activities are integrated by the recruitment of sirtuin 1, a nicotinamide adenine dinucleotide-dependent deacetylase.1,3 The precise roles of acetyltransferases and deacetylases in the transcriptional regulation of FOXO are not known. Recent studies proved that FOXO3a acetylation at three Lys residues (Lys242, Lys245 and Lys259) in the C-terminus of its DNA-binding domain promotes the disruption of protein–DNA contacts and increases the accessibility to AKT phosphorylation at Ser253 thereby attenuating its transcriptional activity.2,4 FOXO3a is involved in the pathogenesis of chronic myeloid leukemia. Its constitutive phosphorylation by p210BCR-ABL tyrosine kinase (TK) through the phosphatidyl-inositol 3-OH kinase/AKT pathway contributes to the proliferative and survival advantage of leukemic progenitors. Accordingly, p210BCR-ABL inhibition by imatinib mesylate (IM, also referred to as STI571 or Glevec) restores FOXO3a functions on cell cycle progression and apoptosis of chronic myeloid leukemic cells through the induction of cyclin-dependent kinase inhibitor p27Kip1 and pro-apoptotic BCL-2L11 BCL-2LIKE 11 (BIM).5,6 Here we investigated the impact of p210BCR-ABL on the acetylation status of FOXO3a. For this purpose, we used a cell clone (3B) generated from 32D murine myeloid progenitor cell line expressing a temperature-sensitive BCR-ABL construct, whose protein possesses TK activity at the permissive temperature of 33 1C, but not at the non-permissive temperature of 39 1C.
First, we confirmed the role of FOXO3a acetylation in signal transduction elicited by growth factor binding to cognate receptors.7 In 32D cells lacking BCR-ABL expression or p210BCR-ABL TK activity (parental cell line and clone 3B at 39 1C, respectively, whose proliferation is strictly growth factordependent), interleukin-3 withdrawal evoked, in fact, FOXO3a acetylation at Lys residues associated with a persistent de-phosphorylation at Ser/Thr residues and prominent nuclear location, and p27Kip1 induction (Figure 1). We then investigated FOXO3a modifications associated with the inhibition of p210BCR-ABL TK, the critical mediator of growth factor independence in leukemic cells. In preliminary experiments (not shown here), we confirmed that in clone 3B kept at 33 1C 1 mM IM abrogates p210BCR-ABL TK activity up to 24 h.8 We found that FOXO3a phosphorylation in the nuclear compartment of clone 3B at 33 1C was significantly reduced from 2 to 8 h of exposure to IM (Figure 2). FOXO3a de-phosphorylation was associated with its increased expression and p27Kip1 induction, supporting its participation in leukemic cell response to IM (Figure 2). However, after longer periods of drug exposure (24 h), nuclear FOXO3a underwent a partial re-phosphorylation associated with hyperacetylation at Lys residues and a significant reduction of p27Kip1 expression (Figure 2). The enhanced nuclear import of acetylated FOXO3a was likely promoted by its progressive acetylation in the cytoplasmic compartment induced, at least in part, by the increased levels and activity of p300 at 2 h
Figure 1 Post-translational modifications of FOXO3a in response to growth factor (interleukin-3 (IL-3)) withdrawal. FOXO3a expression, phosphorylation and acetylation at Lys residues were investigated at 24 h of IL-3 deprivation in nuclear compartments of 32D parental cell line and clone 3B stably transduced with a temperature-sensitive mutant of BCR-ABL kept at the non-permissive temperature (39 1C) for its p210 protein tyrosine kinase activity. They were assayed by labelling the immunoprecipitation products (IP) of FOXO3a with anti-FOXO3a, anti-phospho-Ser/Thr and anti-acetylated Lys antibodies (from Cell Signaling Technology, Trask Lane Danvers, MA, USA). Western blot analysis was used to investigate p27Kip1 expression in the cytoplasm (the anti-p27Kip1 antibody was purchased from Upstate Biotechnology, Lake Placid, NY, USA). Anti-histone H1 and anti-bactin antibodies (from Upstate Biotechnology and Santa Cruz Biotech, Santa Cruz, CA, USA, respectively) were used for protein loading control. Signal intensities from three different blots were quantified by a GS-700 imagining densitometer (Bio-Rad, Waltham, MA, USA) equipped with a dedicated software (Molecular Analyst). Leukemia
Letters to the Editor
406 Acknowledgements This study was supported by University of Bologna, BolognaAIL and Carisbo Foundation. PC and MM are recipients of grants provided by the Istituto di Radioterapia ‘Luigi Galvani’, University of Bologna-Medical School.
P Corrado1, M Mancini1, G Brusa1, S Petta1, G Martinelli1, E Barbieri2 and MA Santucci1 1 Dipartimento di Ematologia e Scienze Oncologiche ‘Lorenzo e Ariosto Sera`gnoli’, University of Bologna-Medical School, Bologna, Italy and 2 Dipartimento di Scienze Radiologiche ed Istopatologiche L Galvani, University of Bologna-Medical School, Bologna, Italy E-mail:
[email protected] References
Figure 2 Post-translational modifications of FOXO3a following p210BCR-ABL tyrosine kinase (TK) inhibition in response to imatinib mesylate (IM). FOXO3a expression, phosphorylation at Ser/Thr residues and acetylation at Lys residues were investigated in the nuclear compartments of clone 3B kept at the permissive temperature for p210BCR-ABL TK (33 1C) at different periods of exposure to IM (1 mM). FOXO3a acetylation at Lys residues in the cytoplasmic compartments of clone 3B was also investigated. p300 levels and AKT activating phosphorylation at Ser473 were assayed by labelling IP with anti-p300 and anti-phospho-Ser473 AKT antibodies (from Upstate Biotechnology and Cell Signaling Technology, respectively). See legend to Figure 1 for technical details.
of exposure to IM (Figure 2; data not shown). FOXO3a acetylation is likely involved in its phosphorylation by AKT, whose enzymatic activity had already recovered at 24 h of IM exposure thereby compensating the persistent inhibition of p210BCR-ABL TK activity (Figure 2).4 In conclusion, our results support FOXO3a acetylation as a late event of response of chronic myeloid leukemia progenitors to IM eventually contributing to the development of a drug-resistant phenotype through mechanisms promoting re-phosphorylation and transcriptional attenuation of FOXO3a.
1 Calnan DR, Brunet A. The FoxO code. Oncogene 2008; 27: 2276–2288. 2 Tsai K-L, Sun Y-J, Huang C-Y, Yang M-C H, Hsiao C-D. Crystal structure of the human FOXO3a-DBD/DNA complex suggests the effects of post-translational modification. Nucleic Acids Res 2007; 35: 6984–6994. 3 Vogt PK, Jiang H, Aoki M. Triple layer control. Phosphorylation, acetylation and ubiquitination of FOXO proteins. Cell Cycle 2005; 4: 908–913. 4 Matsuzaki H, Daitoku H, Hatta M, Aoyama H, Yoshimochi K, Fukamizu A. Acetylation of Foxo1 alters its DNA-binding ability and sensitivity to phosphorylation. Proc Natl Acad Sci USA 2005; 102: 11278–11283. 5 Komatsu N, Watanabe T, Uchida M, Mori M, Kirito K, Kikuchi S et al. A member of forkhead transcription factor FKHRL1 is a downstream effector of STI571-induced cell cycle arrest in BCRABL-expressing cells. J Biol Chem 2003; 278: 6411–6419. 6 Essafi A, Ferna`ndez de mattos S, Hassen YA, Soeiro I, Mufti GJ, Thomas NS et al. Direct transcriptional regulation of Bim by Foxo3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells. Oncogene 2005; 24: 2317–2329. 7 Mahmud DL, G-Amlak M, Deb DK, Platanias LC, Uddin S, Wickrema A. Phosphorylation of forkhead transcription factors by erythropoietin and stem cell factor prevents acetylation and their interaction with coactivator p300 in erythroid progenitor cells. Oncogene 2002; 21: 1556–1562. 8 Mancini M, Brusa G, Zuffa E, Corrado P, Martinelli G, Grafone T et al. Persistent Cdk2 inactivation drives growth arrest of BCR-ABLexpressing cells in response to dual inhibitor of SRC and ABL kinases SKI-606. Leuk Res 2007; 31: 979–987.
High-throughput mutational screen of the tyrosine kinome in chronic myelomonocytic leukemia
Leukemia (2009) 23, 406–409; doi:10.1038/leu.2008.187; published online 10 July 2008
Chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia are characterized by persistent monocytosis and combined dysplastic morphology with features of a myeloproliferative syndrome.1 Together with atypical chronic myelogenous leukemia (aCML) and the category of unclassifiable myelodysplastic/myeloproliferative diseases, they belong to the same WHO diagnostic group.1 Activation of the RAS signaling pathway is almost universal in juvenile myeloLeukemia
monocytic leukemia, albeit the result of diverse genetic lesions, including mutations of N-RAS, K-RAS, NF-1 and PTPN11 (reviewed in Lauchle et al.2). The genetics of CMML and aCML is yet more complex. Various studies reported activating mutations of N-RAS and K-RAS in 20 to almost 60% of patients, and mutations in PTPN11 in isolated cases.3,4 NF-1 (neurofibromatosis 1) has not been studied extensively, but one study of patients with MDS (including CMML) and acute myeloid leukemia did not find major rearrangements at the genomic level.5 In contrast to juvenile myelomonocytic leukemia, activating mutations in tyrosine kinases, including PDGFRA/B, KIT, FGFR1, FLT3, CSF-1R and JAK2, have also been implicated
