Letters to the Editor
abolished B–T-cell cross talk as a result of B-cell depletion in lymphoma patients treated with rituximab. Significantly more pronounced T-cell expansions/imbalances were observed in patients with LON who also exhibited extensive hypoplasia of the granulocytic series with concomitant dyshematopoiesis of moderate-to-severe degree affecting all myeloid series. Therefore, our study demonstrates a remarkable analogy between rituximab-associated LON and neutropenia associated with T-LGL leukemia, which is characterized by BM dyshematopoiesis and/or maturation arrest of the granulocytic series mimicking primary MDS. Of note, BM abnormalities were also evident in rituximab-treated patients without LON, who, in stark contrast to LON cases, were characterized by hyperplasia of all myeloid series albeit again associated with varying degrees of dyshematopoiesis. Taken together, our findings indicate that rituximabassociated LON is multifactorial and may perhaps represent the end of a spectrum of immunohematological sequellae in the context of T-LGL-mediated autoimmune myelopathy/myelodysplasia.
Acknowledgements We thank Mrs Athina Damianaki, Mrs Claudia Gemetzi and Mrs Helen Koutala, at the University of Crete, and Mrs Varvara Tachynopoulou, at the G. Papanicolaou Hospital, for their valuable technical assistance.
K Stamatopoulos1, T Papadaki2, C Pontikoglou3, I Athanasiadou1, N Stavroyianni1, J Bux4, I Batsis1, K Pyrovolaki3, G Paterakis5, D Anagnostou2, A Anagnostopoulos1 and HA Papadaki3 1 Hematology Department and HCT Unit G. Papanikolaou Hospital, Thessaloniki, Greece; 2 Department of Hematopathology, Evangelismos Hospital, Athens, Greece; 3 Department of Hematology, University of Crete School of Medicine, Heraklion, Crete, Greece; 4 Blood Service West of the German Red Cross, Hagen, Germany and
5
Immunology Laboratory, G. Gennimatas Hospital, Athens, Greece E-mail:
[email protected]
1449
References 1 Papadaki T, Stamatopoulos K, Stavroyianni N, Paterakis G, Phisphis M, Stefanoudaki-Sofianatou K et al. Evidence for T-large granular lymphocyte-mediated neutropenia in Rituximab-treated lymphoma patients: report of two cases. Leuk Res 2002; 26: 597–600. 2 Papadaki T, Stamatopoulos K, Anagnostopoulos A, Fassas A. Rituximab-associated immune myelopathy. Blood 2003; 102: 1557–1558. 3 Chaiwatanatorn K, Lee N, Grigg A, Filshie R, Firkin F. Delayed-onset neutropenia associated with rituximab therapy. Br J Haematol 2003; 121: 913–918. 4 Cairoli R, Grillo G, Tedeschi A, D’Avanzo G, Marengo P, Morra E. High incidence of neutropenia in patients treated with rituximab after autologous stem cell transplantation. Haematologica 2004; 89: 361–363. 5 Cattaneo C, Spedini P, Casari S, Re A, Tucci A, Borlenghi E et al. Delayed-onset peripheral blood cytopenia after rituximab: frequency and risk factor assessment in a consecutive series of 77 treatments. Leuk Lymphoma 2006; 47: 1013–1017. 6 Dunleavy K, Hakim F, Kyung Kim H, Janik JE, Grant N, Nakayama T et al. B-cell recovery following rituximab-based therapy is associated with perturbations in stromal derived factor and granulocyte homeostasis. Blood 2005; 106: 795–802. 7 Rezvany MR, Jeddi-Tehrani M, Wigzell H, Osterborg A, Mellstedt H. Leukemia-associated monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic lymphocytic leukemia. Blood 2003; 101: 1063–1070. 8 Liu JH, Wei S, Lamy T, Eppling-Burnette PK, Starkebaum G, Djeu JY et al. Chronic neutropenia mediated by fas-ligand. Blood 2000; 95: 3219–3222. 9 Coakley G, Iqbal M, Brooks D, Panayi GS, lanchbury JS. CD8CD57 T cells from healthy elderly subjects suppress neutrophil development in vitro: implications for the neutropenia of Felty’s and large granular lymphocyte syndromes. Arthritis Rheum 2000; 43: 834–843. 10 Sokol L, Loughran Jr TP. Large granular lymphocyte leukemia. Oncologist 2006; 11: 263–273.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
Inhibition of MEK/ERK signaling synergistically potentiates histone deacetylase inhibitor-induced growth arrest, apoptosis and acetylation of histone H3 on p21waf1 promoter in acute myelogenous leukemia cell
Leukemia (2008) 22, 1449–1452; doi:10.1038/sj.leu.2405079; published online 10 January 2008
Histone deacetylase inhibitors (HDACIs) have emerged as a potentially promising new class of anticancer drugs.1 These include the hydroxamic acid-derived suberoylanilide hydroxamic acid (SAHA, vorinostat), LBH589, tricostatin A, cyclic depsipeptide FR901228 and the benzamide MS-275.2 HDACIs induce growth arrest and apoptosis of cancer cells by manipulating the transcription of genes involved in regulation of the cell cycle and apoptosis, as well as differentiation.1 For example, we previously showed that SAHA induces growth
arrest and apoptosis of human mantle cell lymphoma cells in association with induction of histone acetylation of the p21waf1 promoter region, resulting in upregulation of the p21waf1 protein.3 This study found that MS-275 induces growth arrest of human acute myelogenous leukemia (AML) HL-60 and NB4 cells, as well as freshly isolated leukemia cells from individuals with AML with concentration that induced 50% inhibition (IC50) values less than 1 mM on day 2 of culture, as measured by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and thymidine uptake, respectively (Figure 1a, Table 1). Western blot analyses showed that exposure of these cells to MS-275 downregulated levels of antiapoptotic molecules Bcl-2 and Leukemia
Letters to the Editor
MTT activity (% of control)
1450 120
NB4
100
HL60
80 60 40 20 0 0
0.25
0.5 MS-275 (µM)
HL60 -
0.25
0.75
1
NB4 0.5
1
-
0.25
0.5
1
MS-275 (µM) Bcl-2 Bcl-xL Mcl-1 CyclinD1 p21waf1 ERK p-ERK -tubulin
HL60 -
0.25
0.5
NB4 1
-
0.25
0.5
1
MS-275 (µM) Ac-histone H3 -tubulin
Figure 1 (a) Antiproliferative effects of MS-275 against leukemia cells. The human leukemia HL60 and NB4 cells were plated in 96-well plates and cultured with various concentrations of MS-275 (0.1–1 mM). After 2 days, cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Results represent the mean±s.d. of three experiments performed in triplicate. (b) MS-275 modulates levels of antiapoptotic and cell cycle-related proteins and induces acetylation of histone H3. Western blot analysis. HL60 and NB4 cells were cultured with various concentrations of MS-275 (0.25–1 mM). After 48 h, cells were harvested and subjected to western blot analysis. The membranes were sequentially probed with the indicated antibodies. The figures represent one of the three experiments performed independently. Ac-histone H3, acetylated-histone H3.
Mcl-1, and markedly increased levels of p21waf1 in conjunction with induction of acetylation of histone H3 proteins (Figure 1b). MS-275 also downregulated levels of phospho-extracellular signal regulated kinase (p-ERK) in these cells (Figure 1b). We next explored the drug interaction of MS-275 and AZD6244 (ARRY-142886),4 the specific inhibitor of mitogen-activated protein (MEK)/ERK activity, in AML cells. Interestingly, AZD6244 synergistically potentiated MS-275-induced growth arrest and apoptosis of these cells in association with enhanced induction of p21waf1 (Supplementary Figures 1a–e). We further explored the molecular mechanisms by which blockade of MEK/ ERK signaling enhanced MS-275-induced expression of p21waf1; AZD6244 increased MS-275-induced acetylation of histone H3 proteins in AML cells (Figure 2a). Of note, chromatin immunoprecipitation assay found that combination of Leukemia
AZD6244 and MS-275 synergistically induced acetylation of histone H3 on the p21waf1 promoter in AML cells (Figure 2b). Moreover, MS-275 or AZD6244 either alone modestly downregulated levels of p-histone H3 or combination of both at the same concentration further downregulated levels of p-histone H3 in these cells (Supplementary Figure 2). This is the first observation that blockade of MEK/ERK signaling modulates the epigenetic action of HDACI. Phosphorylation of histone H3 coincided with chromatin condensation in mitosis in eukaryotic cells.2,5 Inhibition of p-histone H3 by this MEK inhibitor may cause chromatin relaxation, leading to susceptibility to HDACIinduced acetylation of the gene promoter. A recent phase I clinical trial has evaluated efficacy and safety of MS-275 in 38 individuals with relapsed or refractory AML. MS-275 induced acetylation of histone H3/H4, expression of
Letters to the Editor
1451 Table 1 Pt no. 1 2 3 4 5 6
Effect of MS-275 on freshly isolated acute leukemia cells Age/sex
FAB
WBC 109
% Blast
67/M 57/M 63/M 79/F 77/F 50/F
M2 M3 M4 M4 M7 M3
38 700 1100 79 400 34 200 2000 6300
54 40 69 50 38 89
Genetic abnormalities t(8;21)(q22;q22) t(15;17)(q22;q12) Normal Normal Complex t(15;17)(q22;q12)
Source
IC50
Previous treatment
PB BM PB PB PB BM
0.05 0.3 0.1 0.06 1 0.06
No No No No Yes No
Abbreviations: BM, bone marrow; F, female; FAB, French–American–British (leukemia classification); M, male; PB, peripheral blood; Pt, patient; WBC, white blood cell. The freshly isolated leukemia cells were cultured in the presence of various concentrations of MS-275 (0.03–1 mM). After 2 days, cell proliferation was measured by tritiated thymidine uptake. The results were graphed and the concentration of MS-275 that induced 50% inhibition (IC50) of thymidine uptake was calculated from the dose–response curves.
NB4
HL60 -
+ -
+
+ +
-
+ -
+
+ +
MS-275 AZD6244 Ac-histone H3 -tubulin
HL60 -
+ -
NB4 +
+ +
-
+ -
+
+ +
MS-275 AZD6244
ChIP
p21waf1 promoter
Input
p21waf1 promoter
Without ChIP
p21waf1 promoter
Figure 2 AZD6244 enhanced MS-275-induced acetylation of histone H3. (a) Western blot analysis. HL60 and NB4 cells were cultured in the presence of either MS-275 (0.5 mM) and/or AZD6244 (0.25 mM). After 6 h, cells were harvested and nuclear proteins were prepared and subjected to western blot analysis. The membranes were sequentially probed with anti-acetyl-histone H3 and anti-a-tubulin antibodies. The figure is representative of three similar experiments. Ac-histone H3, acetylated-histone H3. (b) Chromatin immunoprecipitation assay. Acetylation of histone H3 in the p21waf1 promoter was analyzed by chromatin immunoprecipitation assay. HL60 and NB4 cells were cultured in the presence of either MS-275 (0.5 mM) and/or AZD6244 (0.25 mM). After 6 h, cells were harvested and subjected to chromatin immunoprecipitation assay. Anti-acetylated histone H3 antibody or rabbit immunoglobulin G (IgG) was used to immunoprecipitate soluble chromatin from these cells. The recovered DNAs were subjected to PCR using primers for p21waf1promoter. The result is representative of two additional experiments performed independently.
p21waf1 and activation of caspase 3 in bone marrow mononuclear cells, although no responses were observed by classical criteria.6 Taken together, combination of MS-275 and AD6244 may be useful for treatment of individuals with AML. Further studies are required to verify the molecular mechanisms by which blockade of MEK/ERK signaling affects chromatin remodeling.
Acknowledgements This work was supported in part by grant-in-aid from the Ministry of Education, Culture Sports, Science, and Technology of Japan; Kanae Foundation for the Promotion of Medical Science; Public Trust of Haraguchi Memorial Cancer Research Fund and the Fund for Academic Research from Kochi University. HPK is supported by NIH grants, as well as the Inger Fund and the Parker Hughes Trust.
C Nishioka1,3, T Ikezoe1, J Yang1, HP Koeffler 2 and A Yokoyama1 1 Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan and 2 Department of Hematology and Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA, USA E-mail:
[email protected] 3 The author is a research fellow of the Japanese Society for the Promotion of Science (JSPS), Japan.
References 1 Minucci S, Pelicci PG. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 2006; 6: 38–51. Leukemia
Letters to the Editor
1452 2 Dyson MH, Thomson S, Inagaki M, Goto H, Arthur SJ, Nightingale K et al. MAP kinase-mediated phosphorylation of distinct pools of histone H3 at S10 or S28 via mitogen- and stress-activated kinase 1/2. J Cell Sci 2005; 118: 2247–2259. 3 Sakajiri S, Kumagai T, Kawamata N, Saitoh T, Said JW, Koeffler HP. Histone deacetylase inhibitors profoundly decrease proliferation of human lymphoid cancer cell lines. Exp Hematol 2005; 33: 53–61. 4 Nishioka C, Ikezoe T, Takeshita A, Yang J, Tasaka T, Yang Y et al. ZD6474 induces growth arrest and apoptosis of human leukemia
cells, which is enhanced by concomitant use of a novel MEK inhibitor, AZD6244. Leukemia 2007; 21: 1308–1310. 5 Prigent C, Dimitrov S. Phosphorylation of serine 10 in histone H3, what for? J Cell Sci 2003; 116: 3677–3685. 6 Gojo I, Jiemjit A, Trepel JB, Sparreboom A, Figg WD, Rollins S et al. Phase 1 and pharmacological study of MS-275, a histone deacetylase inhibitor, in adults with refractory and relapsed acute leukemias. Blood 2007; 109: 2781–2790.
Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
Tandutinib inhibits FMS receptor signalling, and macrophage and osteoclast formation in vitro targets the Flt-3 receptor.1 Flt-3 is constitutively activated by internal tandem duplications in up to 30% of acute myelogenous leukemia patients, and a further 5–10% of patients have oncogenic mutations of Asp835 of the kinase domain.2 Flt-3 is closely related to the KIT, PDGF and FMS receptors, and both KIT and PDGF receptors are targets of tandutinib with IC50
Leukemia (2008) 22, 1452–1453; doi:10.1038/sj.leu.2405085; published online 10 January 2008
Tandutinib (MLN518; CT53518) is a promising quinazolinebased tyrosine kinase inhibitor that is currently in phase II clinical trails for treatment of acute myeloid leukaemia, where it
Cell Number (cell/ml)
IL-3
Wt FMS FMS D802V
4000000
tandutinib (µM) M-CSF
0 -
0 +
0.25 0.5 + +
1 +
2 +
FMS (Y708P)
3000000 FMS 2000000 tandutinib (µM)
1000000
0
0.25
0.5
1
2
FMS (Y723P)
0 0 0.1 0.2 0.4 0.8 1.6 3.2
tandutinib (µM)
FMSD802VY708F
tandutinib (µM)
0
0.125 0.25 0.5
1
2
FMS kinase assay
18 16 14 12 10 8 6 4 2 0
tandutinib Imatinib
M-CSF 0.5µM
1µM -M-CSF
% FMS activity
Colony number
FMSD802VY708F Kinase assay WT FMS FMS D802VY708F
120 100 80 60 40 20 0
1
2
3 4 Lanes
5
6
Figure 1 Tandutinib inhibits FMS receptor signalling, osteoclastogenesis and macrophage colony formation. (a) FDC-P1 haematopoietic cells that express FMS were seeded at 5 104 cells in either 10 ng ml1 IL-3 (diamonds) or 5000 U ml1 macrophage colony-stimulating factor (M-CSF; circles) with the indicated concentrations of tandutinib. Similarly, FDC-P1 cells that express the oncogenic receptor FMS D802V were cultured with tandutinib in the absence of both M-CSF and IL-3 (triangles). Viable cell numbers were counted 5 days later. (b, c) Rat2vfms stained with Geimsa following growth in the absence (b) or presence of 1 mM tandutinib (c). (d) Mouse bone marrow-derived macrophages were cultured in methylcellulose with 5000 U ml1 M-CSF, and macrophage colonies were counted after 2 weeks and are shown as colony count per 5 104 bone marrow mononuclear cells seeded. (e, f) Rat-2 fibroblasts expressing wild-type FMS (e) or FMS D802VY708F (f) were pretreated with the indicated concentrations of tandutinib for 1 h and stimulated with M-CSF (5000 U ml1) for 5 min prior to immunoblotting with antibodies to phospho-FMS tyrosine723 (e, f upper panel) or FMS (e, f lower panel). (g) In vitro kinase assay. FMS or FMSD802VY708F receptors were immunoprecipitated from Rat-2 fibroblasts with a rabbit polyclonal antibody and pretreated with the indicated concentrations of tandutinib for 5 min at room temperature and then incubated with 32P-labelled gATP for 15 min at 30 1C. Kinase activity was measured by SDS-PAGE (polyacrylamide gel electrophoresis) autoradiography, and densitometry readings are shown for FMS (diamonds) and FMSD802VY708F (squares). Leukemia
