Apoptosis (2008) 13:1148–1161 DOI 10.1007/s10495-008-0243-4
ORIGINAL PAPER
Flt3 receptor inhibition reduces constitutive NFjB activation in high-risk myelodysplastic syndrome and acute myeloid leukemia Jennifer Grosjean-Raillard Æ Lionel Ade`s Æ Simone Boehrer Æ Maximilien Tailler Æ Claire Fabre Æ Thorsten Braun Æ Ste´phane De Botton Æ Alain Israel Æ Pierre Fenaux Æ Guido Kroemer Published online: 1 August 2008 Ó Springer Science+Business Media, LLC 2008
Abstract High-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are characterized by the activation of the anti-apoptotic transcription factor NFjB, via the IKK complex. Here, we show that constitutive activation of the receptor tyrosine kinase Flt3 is responsible for IKK activation. Chemical inhibition or knockdown of Flt3 with small interfering RNAs reduced NFjB activation in MDS and AML cell lines, as well as in
Author contributions: J.G-R. performed the experiments and analyzed the data. L.A., S.B., C.F., T.B. and S.d.B. provided bone marrow samples and essential clinical information on patients. A.I and P.F. participated in the conception of the study. G.K. conceived and directed the study. J.G-R and G.K. wrote the paper.
Electronic supplementary material The online version of this article (doi:10.1007/s10495-008-0243-4) contains supplementary material, which is available to authorized users. J. Grosjean-Raillard L. Ade`s S. Boehrer M. Tailler C. Fabre G. Kroemer (&) INSERM, U848, Institut Gustave Roussy, PR1, 38 rue Camille Desmoulins, 94805 Villejuif, France e-mail:
[email protected] J. Grosjean-Raillard L. Ade`s S. Boehrer M. Tailler C. Fabre S. De Botton G. Kroemer Institut Gustave Roussy, 94805 Villejuif, France J. Grosjean-Raillard L. Ade`s S. Boehrer M. Tailler C. Fabre Universite´ Paris Sud, Paris 11, 94805 Villejuif, France L. Ade`s S. Boehrer T. Braun P. Fenaux Service d’He´matologie Clinique, Hoˆpital Avicenne, AP-HP, Universite´ Paris XIII, Bobigny, France A. Israel Unite´ de Signalisation Moleculaire et Activation Cellulaire, URA 2582 CNRS Institut Pasteur, 75015 Paris, France
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primary CD34+ bone marrow cells from patients, causing apoptosis. Epistatic analysis involving the simultaneous inhibition of Flt3 and IKK suggested that both kinases act in the same anti-apoptotic pathway. An IKK2 mutant with a constitutive kinase activity and a plasma membranetethered mutant of NEMO that activates IKK1/2 prevented the cytocidal action of Flt3 inhibition. Flt3 phosphorylates IKK2 in vitro, and Flt3 inhibition reduced the phosphorylation of IKK2 in MDS or AML cell lines. IKK2 and Flt3 physically associated in MDS and AML cells, and Flt3 inhibition disrupted this interaction. Flt3 inhibition only killed CD34+ bone marrow cells from high-risk MDS and AML patients, in correlation with blast numbers and NFjB activity, yet had no lethal effect on healthy CD34+ cells or cells from low-risk MDS. These results suggest that Flt3 inhibitors might exert an anti-neoplastic effect in high-risk MDS and AML through inhibition of NFjB. Keywords IKK
Acute myeloid leukemia Flt3 NFjB
Abbreviations Flt3 Fms-like tyrosine kinase-3 Flt3I Flt3 inhibitor AML Acute myeloid leukemia BMMNC Bone marrow mononuclear cells NFjB Nuclear factor-jB DAPI 40 ,6-diaminidino-2-phenylindole DiOC6(3) 3,30 dihexyloxacarbocyanine iodide GAPDH Glyceraldehyde-3-phosphate dehydrogenase IjB Inhibitor of NFjB IKK IjB kinase MDS Myelodysplastic syndrome
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PI Z-VAD.fmk
Propidium iodide N-benzyloxycarbonyl-Val-AlaAsp-fluoromethylketone
Introduction Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress into acute myeloid leukaemia (AML). A plethora of genetic aberrations have been detected in patients with MDS, and some of these genetic alterations are associated with the progression of MDS to leukemia. Activating mutations of the receptor tyrosine kinase fmslike tyrosine kinase 3 (Flt3), a member of the class III receptor tyrosine kinase family, are the most common tyrosine kinase mutations in acute myeloid leukemia (AML). Flt3 is highly expressed in most patients with AML and approximately 30% of these patients have activating mutations in Flt3. Nearly 30% of AML patients possess an internal tandem duplication (ITD) within the juxtamembrane domain and an additional 7% have point mutations such as the D835Y mutation within the catalytic domain of the kinase [1, 2]. Moreover, increased levels of Flt3 transcripts are observed in a large number of AML patients and this increased expression may also contribute to the phosphorylation and activation of Flt3 [3]. Notably, more than two-thirds of AML patients exhibit the (auto-) phosphorylation of Flt3 on tyrosine residues 589 and 591 even in the absence of activating mutations [4]. Both types of mutation result in constitutive activation of Flt3 leading to activation of downstream signaling pathways, giving cells a proliferative and survival advantage [5–7]. Flt3 phosphorylation/activation results in the activation of downstream kinase pathways, like the Ras/mitogen-activated protein kinase (MAPK), allowing abnormal cell growth and aberrant gene regulation [8–10]. Only a few studies have analyzed Flt3 alterations in patients with MDS and suggest that Flt3 mutations are rare in MDS, affecting 5–10% of high-risk MDS [1, 5, 11]. Several master regulators of proliferation and apoptosis are transactivated by the nuclear factor kappa B (NFjB) [12, 13], a transcription factor that is often activated in neoplasia. It has been reported that, in most if not all cases of AML, NFjB is constitutively activated and its target genes are upregulated [14]. Recently, it has been reported that NFjB can be activated via the Ras/phosphatidylinositol-3-kinase (PI3K)/protein kinase B pathway in AML [15]. NFjB is also constitutively active in high-risk MDS (with high or int-2 IPSS scores), yet inactive in low risk MDS (with low or int-1 IPSS) [16]. This correlates with increased spontaneous apoptosis inhibition in high-risk
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MDS myeloblasts. Inhibition of NFjB can trigger apoptosis in high-risk MDS as well as in AML [17]. NFjB activation depends on phosphorylation and degradation of inhibitor proteins collectively termed IjB, which sequester NFjB in the cytoplasm. Stimulation of the NFjB pathway leads to the rapid phosphorylation of IjBs and consequent degradation, resulting in NFjB translocation into the nucleus. NFjB molecules exist as dimers of the ‘rel’ protein family, whose members include p50/p105 (NFjB1), p52/100 (NFjB2), p65 (RelA), RelB and c-Rel. Members of the rel family are characterized by a conserved N-terminal 300 amino acid sequence, the rel homology domain. This domain enables dimerisation, DNA binding, nuclear localisation and interaction with the IjB family of proteins. The phosphorylation of IjBa on serine residues 32 and 36 is initiated by an IjB kinase (IKK) complex which, at least in the canonical NFjB activation pathway, includes a catalytic heterocomplex composed of IjB kinase (IKK)-1, IKK-2 and NEMO [12, 13, 18, 19]. According to one study, Flt3 activation by addition of the Flt3 ligand (Flt3L) can trigger NFjB activation [20]. Stimulated by these report, we investigated whether inhibition of Flt3 might inhibit NFjB activation in high-risk MDS and AML. To date, several different Flt3 kinase inhibitors have been developed and tested in vitro [21–23] and in early clinical trials with variable degrees of success [24–26]. Up to 80% of patients in these studies achieved blast reduction over a few months. Another approach for targeting Flt3 involves RNA interference (siRNA), which induced down regulation of Flt3 expression [27]. Thus, the aim of this study was to examine whether Flt3 depletion or inhibition would induce cell death through an effect on the NFjB pathway and, if so, through which molecular mechanisms.
Material and methods Patients AML, MDS patients and healthy subjects were included in our study (Table 1). Informed consent of all patients and healthy subjects was provided according to the Declaration of Helsinki. This study was approved by the Institut Gustave Roussy institutional review board. MDS patients were previously untreated except for supportive care. The diagnosis of MDS was based on peripheral blood counts, cytology of peripheral blood and bone marrow (BM) according to the WHO classification [28] and conventional cytogenetic analysis. These data evaluation of the individual International Prognostic Scoring System (IPSS) score for each patient [16]. BM aspirates were collected, after informed consent, into syringes containing media
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supplemented with EDTA. The BM mononuclear cell (BMMNC) fraction was isolated by density gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Sunnyvale, CA, USA) and then washed three times (10 min, 600g, 4°C) in complete medium. Cells and culture conditions The MDS cell line MOLM-13 and the AML cell lines MV4-11 and KG1 were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany. All cell lines were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/ml penicillin and 100 lg/ml streptomycin as described [29]. BM cells were isolated and processed as follows: after density gradient (Ficoll-Paque Plus) separation of BM mononuclear cells (BMMNC), CD34+ cells were isolated by positive selection with the MiniMacs system (Miltenyi Biotec, Bergish Gladbach, Germany) and then maintained in Iscove’s modified Dulbecco’s medium (Gibco) supplemented with 10% heat-inactivated FCS. We used distinct inhibitors to suppress the enzymatic activity of Flt3, in particular the Flt3 inhibitor 2-acylaminothiophene-(3,4-di-O-methylphenyl)-3-carboxamide (Flt3I) (Calbiochem, Darmstadt, Germany). To induce Flt3 activation in KG-1 cell, Flt3 ligand (Fl3L, Calbiochem) was used overnight at a concentration of 10 lg/ml. Transfection of plasmids and knockdown of Flt3 by siRNA
Apoptosis (2008) 13:1148–1161
(Becton Dickinson, Mountain View, CA, USA) as described previously [31, 32]. Cells were stained with propidum iodine (PI, 10 lM, Sigma, St Louis, MO, USA) and 20 nM fluorochrome DiOC6(3) (Molecular Probes, Eugene, OR, USA) for 15 min at 37°C, for the determination of plasma membrane permeability and the mitochondrial transmembrane potential (DWm), respectively [33, 34]. Nuclear protein extraction and electrophoretic mobility shift assay Nuclear extracts were prepared from all cell lines after culture in the presence or absence of Flt3 inhibitor (standard treatment: 2 lM, 2 h). Cells were harvested and washed twice with ice-cold phosphate-buffered saline (PBS). Cell pellets were then lysed in hypotonic lysis buffer (10 mM N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid-potassium hydroxide (HEPES), 1.5 mM MgCl2, 10 mM KCl and 0.0125% NP-40, pH 7.9). After incubation on ice for 10 min, nuclei were separated from the cytosolic extracts by centrifugation (13,000g, 5 min at 4°C). The nuclear pellets were resuspended in hypertonic extraction buffer (5 mM HEPES, 1 mM MgCl2, 0.2 mM ethylenediaminetetra-acetic acid (EDTA), 0.5 M NaCl, 25% glycerol, and 0.025% NP-40, pH 7.0) for 30 min at 4°C under agitation. After centrifugation at 13,000g, 15 min at 4°C), supernatants containing nuclear proteins were removed and stored at -70°C. Nuclear extracts were examined for NFjB-binding activity by EMSA, using a non-radioactive EMSA kit (Panomics, Freemont, CA, USA). Immunofluorescence stainings
Cells were transfected with the Nucleofactor system (Amaxa, Cologne, Germany) using siRNAs specific for GFP (50 -GCAAGCTGACCCTGAAGTTCA-30 ) with no homology to human genes (EGFP siRNA; InvivoGen, San Diego, USA), a pool of siRNAs specific for Flt3 (Upstate, Lake Placid, NY, USA), two individual validated Flt3 siRNAs (Hs_Flt3_6 HP and Hs_Flt3_7 HP QIAGEN, Valencia, California, USA), siRNAs specific for p65, IKK1, IKK2 or NEMO (described in Ref. [30]) and/or plasmids VSV-IKK2 h, Flag-IKK2hSS[EE, Flag-NEMOm, Myr-NEMOm(MN)40, [31]. Cells were used 48 h after transfection. Assessment of apoptosis Cells (105) from all MDS and AML cell lines or patient cells were resuspended in 1 ml of culture medium and incubated in the presence or absence of 2 lM Flt3I and/or 50 lM Z-VAD-fmk, for 48 h. Apoptotic cells were detected by flow cytometric analysis using a FACScan
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Cell lines or patient cells (105) were allowed to adhere to polylysin-L coverslips (Sigma) and fixed in PBS containing 4% paraformaldehyde at room temperature. Cells were then permeabilized either with 0.05% Triton X-100 (Boehringer, Mannheim, Germany) or 0.1% sodium dodecyl sulfate (SDS) for 10 min, washed in PBS and stained with antibodies specific for p65 (rabbit polyclonal Ab, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and revealed with a goat anti-rabbit IgG coupled with Alexa 568 (Molecular Probes). Nuclei were counterstained with DAPI (Molecular Probes) allowing for the discernment of chromatin condensation [31]. 200 cells for each slide were examined independently with a LSM 510 confocal microscope (Zeiss) at 63-fold magnification. Immunoblots Cellular lysates (50 lg protein/lane) were subjected to SDS/polyacrylamide gel electrophoresis, transferred on
78
70 76
24
25 26
85
70
23
27
55
86
14
87
87
13
22
85 58
11 12
21
69
10
56
72
9
20
56
8
61 83
77
7
18 19
78
6
84
55
5
17
57
4
69
63
3
67
59
2
15
47
1
16
Age
Number
47,XY, +8
46,XX 46,XY
46,XY, del(5q)
46,XX
45,XY, del(20q)
46,XX
46,XY
46,XY 46,XY, del(12p)
46,XX, del(11q)
46,XX
46,XY
46,XY
46,XY, del(20q)
46,XY 46,XY
Failure
46,XY
46,XY
46,XX
46,XX
46,XY
46,XY
46,XX
46,XX
46,XX
Karyotype
RAEB-2
RAEB-2 RAEB-2
RAEB-2
RAEB-2
RAEB-2
RAEB-2
RAEB-2
RAEB-1 RAEB-2
RAEB-1
RAEB-1
CMML
CMML
RCMD
RARS RCMD
RAEB-1
Pure RA
Pure RA
Pure RA
Pure RA
Control
Control
Control
Control
Control
Disease
High
INT-2 INT-2
INT-2
INT-2
INT-2
INT-2
INT-2
INT-2 High
INT-2
INT-2
INT-2
INT-1
Low
Low Low
NA
INT-1
Low
Low
Low
MDS IPSS score
Table 1 Characteristics of MDS patients with nuclear NFjB analysis
High-risk MDS
Low risk MDS
Control
16
11 16
16
11
11
12
15
13 13
11
15
11
1
2
0 1
7
0
0
0
0
0
0
0
0
0
% Bone marrow blasts
+
+
+
+
-
+
+
+ ND
+
-
+
-
-
+ +
ND
ND
-
-
-
-
-
-
-
-
Phospho-Flt3 immunostaining
66
26 65
70
24
10
17
18
76 14
56
23
19
58
9
32 84
8
5
2
3
0
0
0
0
0
0
% Nuclear NFjB staining without Flt3I
18
18 16
26
17
10
8
5
21 6
3
19
9
17
8
2 53
4
2
2
3
0
0
0
0
0
0
% Nuclear NFjB staining with Flt3I
48
41 35
37
48
49
56
65
57 59
63
72
70
56
55
62 64
68
71
76
73
75
10
8
12
17
21
% Apoptosis without Flt3I
63
64 65
63
60
69
60
71
75 70
74
80
81
65
58
65 75
76
78
82
92
84
9
15
25
22
21
% Apoptosis with Flt3I
Apoptosis (2008) 13:1148–1161 1151
123
sAML
sAML
46,complex
46,XY 36
78
sAML 46,XY, del(5q)
35
34
85
AML 47,XY, +21 33
62
AML 48,XY, +8+21 32
42
AML 46,XY 31
41
AML 46,XY, inv(16)
66
AML 46,XY 70
36 30
29
28
RA, refractory anemia; RARS, RA with ring sideroblasts; RCMD, efractory cytopenia with multilineage dysplasia; CMML, chronic myelomonocytic leukaemia; RAEB, refractory anemia with excess of blasts (5–9% BM blasts RAEB-1, 10–19% BM blasts RAEB-2); sAML, secondary aML; ND: not determined
58
56
23 25 31 54
ND
21
60 28
32
29
84
-
50
33
+
22
80
63 25 48 30
ND
31
74
45 59 50
+
11
86
44 22 82 90
+
50
82 65
11 89
17 38
+ 68
55
-
54 8 64 AML 46,XY
123
56
Disease Karyotype Age Number
Table 1 continued
MDS IPSS score
AML
70
+
% Apoptosis without Flt3I % Nuclear NFjB staining without Flt3I % Bone marrow blasts
Phospho-Flt3 immunostaining
% Nuclear NFjB staining with Flt3I
88
Apoptosis (2008) 13:1148–1161 % Apoptosis with Flt3I
1152
nitrocellulose membranes and immunochemical detection was carried out using antibodies specific for IjBa (rabbit polyclonal Ab, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Phospho-IKK1-2 (Cell Signaling Technology Boston, Massachusetts, USA), IKK1 (rabbit polyclonal Ab, Santa Cruz Biotechnology, Santa Cruz, CA, USA), IKK2 (rabbit polyclonal Ab, Santa Cruz Biotechnology, Santa Cruz, CA, USA), NEMO (rabbit polyclonal Ab, Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphotyrosine (Cell Signaling Technology, Boston, Massachusetts, USA) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, Massachusetts, USA). Immunoprecipitation Following treatment, cells were lysed in RIPA buffer (50 mM Tris HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and 200 lg protein were immunoprecipitated with the antibody of interest for 2 h and incubated with protein G-coupled sepharose beads (Roche Applied Science, Rockford, llinois, USA) overnight at 4°C. Immunoprecipitates were obtained following several centrifugation/wash cycles in lysis buffer followed by immunoblotting. Flt3 kinase assay An IKK2-derived peptide was purchased from Merck (IKKb, HisTagÒ, Merck Chemicals Ltd, Nottingham, UK) and was biotinylated using EZ-LinkTM Sulfo-NHS-Biotin according to manufactures instruction (Pierce, Rockford, Illinois, USA). Biotinylated peptide was then evaluated for phosphorylation by Flt3 using a specific assay kit (Cell Signalling Technology for Perkin Elmer-Delfia, Boston, Massachusetts, USA). In a total reaction volume of 50 ll, 100 ng recombinant Flt3 kinase, 1.5 lM biotinylated peptide, 20 lM ATP and variable amounts of Flt3I were␣allowed to react at room temperature for 30 min. The reaction was then stopped and samples loaded on a streptavidin coated plate. The phosphorylation of biotinyated-IKK2 peptide by Flt3 kinase was detected using a phospho-tyrosine-specific mAb (P-Tyr-100), and Europium-labeled secondary antibody (Cell Signalling Technology for Perkin Elmer-Delfia, Boston, Massachusetts, USA), and the measurement of European-dependent fluorescence (excitation at 340 nm and emission at 615 nm).
Results Pharmacological inhibition of Flt3 also inhibits NFjB and induces apoptosis in MDS and AML cell lines. We chose to investigate the effects of Flt3 inhibition on two distinct
Apoptosis (2008) 13:1148–1161
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cell lines that both express active, phosphorylated Flt3 (on tyrosines 589/591), namely the MOLM-13 cell line (which is derived from a patient with high-risk MDS) [2, 35] and the MV4-11 cell line (which is derived from a patient with
AML) [2] (Fig. 1a). When Flt3 was immunoprecipiated from MOLM-13 cells (Fig. 1b) or MV4-11 cells (not shown), the protein was found to react with an antibody recognizing phosphotyrosine residues irrespective of their
Fig. 1 Apoptosis induction by Flt3 inhibition in MOLM-13 and MV4-11 cells. (a) Immunoblot detection of Flt3 and its activating phosphorylation in different cell lines. Extracts from the indicated cell lines were subjected to SDS PAGE and immunoblot detection of Flt3 and GAPDH as a loading control. Note the presence of a band with a reduced electrophoretic mobility in MOLM-13 and MV4-11 cells. (b) Tyrosine phosphorylation of Flt3 in MOLM-13 cells. Flt3 was immunoprecipated and the immunoprecipiated was immunoblotted for the detection of Flt3, phosphotyrosine residues and phospho-Flt3, as described in Materials and Methods. Whole cell lysates and an immunoprecipitation with non-specific rabbit IgG were included as controls. (c, d). Detection of mitochondrial signs of apoptosis after treatment with Flt3I. MOLM-13 or MV4-11 cells were cultured in the
absence or presence of Flt3I (2 lM) and/or Z-VAD-fmk (50 lM) for 18 h and then stained with the DWm-sensitive dye DiOC6(3) and the vital dye propidium iodide (PI), followed by cytofluorometric analysis. The percentage (X ± SD of triplicates) of dying (DiOC6(3)low PI-) or dead (DiOC6(3)low PI-) cells are represented for MOLM-13 cells in B and for MV4-11 cells in C. (e–g) Detection of apoptotic DNA loss after Flt3 inhibition (2 lM, 18 h). Cells were ethanol-fixed, treated with RNAse, labeled with PI and subjected to cell cycle analysis. Representative FACS histograms are shown in d (bars indicate the gate containing subdiploid cells) for MOLM-13 cells cultured as in b. Quantitative data (X ± SD of triplicates) are represented for MOLM-13 cells in e and for MV4-11 cells in f
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Fig. 2 Effect of Flt3 inhibition on the NFjB system in MOLM-13 and MV4-11 cells. (a, b) Detection of activating phosphorylations of Flt3 (on tyrosines 589/591), IKK1/2 (serine 176 or 180), and IjBa degradation after addition of Flt3I (2 lM, 2 h) to the indicated cell types. (c, d) Inhibition of NFjB by Flt3, as determined by different techniques in MOLM-13 and MV4-11 cells. The abundance of NFjB
in nuclear extracts prepared from untreated or Flt3-inhibited (2 lM, 2 h) was determined by EMSA (c). Alternatively, the presence of p65 in nuclei was determined by immunofluorescence (d). Percentages refer to the frequency of cells that contain NFjB in their nuclei. All experiments were repeated at least three times, yielding similar results
surrounding peptide sequence, as well as with an antibody that specifically recognizes phosphorylated Flt3 (on tyrosines 589/591) (Fig. 1b). Addition of the Flt3 inhibitor 2acylaminothiophene-(3,4-di-O-methylphenyl)-3-carboxamide (Flt3I) [36] induced signs of apoptosis in both cell lines, namely a loss of the mitochondrial transmembrane potential (DWm, determined by staining with the DWm-sensitive dye DiOC6 (3)) that preceded the loss of viability (determined with the vital dye propidium iodide, PI) (Fig. 1c, d) and DNA degradation leading to an accumulation of cells with a subdiploid DNA content (Fig. 1e–g). Addition of the pan-caspase inhibitor Z-VAD-fmk had a minor effect on these apoptosis-associated parameters, suggesting that they occurs in a mostly caspase-independent fashion (Fig. 1e–g). MOLM-13 and MV4-11 cells exhibit constitutive activation of the NFjB pathway, and inhibition of NFjB kills these cells efficiently [30]. In both MOLM-13 and MV4-11 cells, Flt3I greatly reduced the activating phosphorylation of Flt3 (Fig. 2a), as well as the phosphorylation of the inhibitor of NFjB kinase (IKK) subunits 1/2 (IKK1/2), as observed using a phospho-neoepitope-specific antibody. Flt3I also enhanced the abundance of the IKK substrate, inhibitor of NFjB (IjBa) (Fig. 2b). Concomitant with the inhibition of IKK1/2 and IjB degradation, Flt3I reduced the nuclear presence of nuclear NFjB, as determined by by electrophoretic mobility shift assays (Fig. 2c) or immunofluorescence detection of the p65 NFjB subunit (Fig. 2d),
in both MOLM-13 and MV4-11 cells. Again, these effects were rapid, leading to strong NFjB inhibition within 1–2 h (Fig. 2c, d). Since chemical inhibitors of tyrosine kinases often (if not always) inhibit other targets than the kinase that they had originally been designed for, we attempted to exclude possible off-target effects of Flt3I by an alternative strategy of Flt3 inhibition. Knockdown of Flt3 with a pool of specific siRNAs (which was maximal 48 h after transfection) reduced the phosphorylation of IKK1/2 and increased the abundance of IjBa (Fig. 3a), as well as that of EMSAdetectable NFjB in the nucleus (Fig. 3b), while causing cell death (Fig. 3c). Note that the depletion of Flt3 was near to complete (Fig. 3) when still 30–40% of the cells were viable (Fig. 3f), indicating that the disappearance of immunodetectable Flt3 cannot be explained by massive cell death. Similar results were obtained when Flt3 was depleted by two distinct individual siRNAs, resulting in reduced IKK1/2 phosphorylation, enhanced IjBa expression as well as cell death (Fig. 3d–f). Altogether, these results indicate that Flt3 knockdown can inhibit the IKKdependent activation of NFjB and stimulate apoptosis in cell lines that express constitutively active Flt3. In a well characterized AML cell line, KG1 (which does not manifest any activating phosphorylation of Flt3, Fig. 1a), inhibition of Flt3 had no pro-apoptotic activity. Conversely, addition of the Flt3 ligand Flt3 reduced the spontaneous cell death of KG1 cells (Supplementary
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Fig. 3 Knockdown of Flt3 causes NFjB inhibition and apoptosis. (a–c) Effect of a pool of Flt3-specific siRNAs. MOLM-13 and MV4-11 cells were electroporated with the indicated siRNA pool, and the expression level and phosphorylation status of Flt3, IKK1/2 and degradation of IjBa were detected by immunoblot 48 h later (a). NFjB activation was measured by EMSA (b) and the frequency of dying cells (X ± SD, n = 3) was determined by DiOC6(3)/PI staining (c). (d–e) Effect of individual Flt3-specific siRNAs. MOLM-13 and MV4-11 cells were electroporated with 2 distinct Flt3 siRNA and the expression level Flt3 was detected by immunoblot 48 h later (d), NFjB activity was measured by EMSA (f) the frequency of dying cells (X ± SD, n = 3) was determined by DiOC6(3)/PI staining (e)
Fig. 1a). In these cells, which do not activate NFjB constitutively, ligation of Flt3 with Flt3 enhanced the degradation of IjBa (Supplementary Fig.1b) and activated
NFjB (Supplementary Fig. 1c, d). Taken together, these data suggest that activation of Flt3 is intimately linked to anti-apoptotic NFjB activation.
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Fig. 4 Epistatic and functional analysis of the cause-effect relationship between apoptosis induction and IKK inhibition by Flt3 depletion. (a) Knockdown of distinct subunits of the IKK complex by specific siRNAs, as determined 48 h after electroporation of MOLM-13 cells. (b) Epistatic analysis of apoptosis induction by depletion of Flt3. Cells were transfected with the indicated combination of siRNAs (using either the siRNAs used in A or a pool of Flt3-specific siRNAs used in Fig. 3a–c), and cell death (X ± SD, n = 3) was measured by DiOC6(3)/PI staining and cytofluorometry. (c) Transfection-enforced overexpression of wild type (WT) active mutants of IKK2 (CA-IKK2) and NEMO (NEMO-NM). MOLM-13 cells were transfected with vector-only or the indicated cDNAs, and
protein expression was monitored 48 h after transfection. (d) Inhibition of the cytocidal effects of Flt3 knock-down by active IKK2 and NEMO. Cells were simultaneously transfected with control siRNA or FLT-R-specific siRNA and/or the indicated IKK2 and NEMO constructs. Two days later the frequency of dead and dying cells was assessed by the DiOC6(3)/PI method. (e) Abolition of Flt3Imediated killing by activation of the IKK complex. Cells were first transfected with the indicated control constructs and normal or active versions of IKK2 or NEMO. Twenty-four h later the cells were treated for further 18 h with Flt3I, followed by cell death assessment. All results are means ± SD of triplicates of one experiment representative of three or more
Inhibition of Flt3 induces apoptosis through IKK inhibition. As shown above, pharmacological Flt3 inhibition or Flt3 knockdown causes apoptosis and inhibits NFjB. To establish a cause-effect relationship between cell death and NFjB inhibition, we employed three complementary strategies. First, we performed an epistatic analysis of the proapoptotic effects of Flt3 depletion with simultaneous inhibition of different components of the NFjB activation pathway. MOLM-13 cells were transfected with siRNAs that efficiently targeted Flt3, IKK1, IKK2 or NEMO (Fig. 4a), alone or in combination. Both the depletion of Flt3 and that of any of the component of the IKK complex (IKK1, IKK2, NEMO) induced cell death. However, combined Flt3 and IKK depletion was no more efficient
than the inhibition of Flt3 or IKK alone (Fig. 4b), suggesting that Flt3 and IKK act in the same pathway. Second, we transfected MOLM-13 cells with a constitutive active (CA) form of IKK2 (due to two point mutations in the activation loop, S177E and S181E, increasing the kinase activity of the protein) [37] or wild type (WT) IKK2 as a control. Alternatively, the cells were transfected with a version of NEMO that activates the IKK complex (due to introduction of an N-terminal myristoylation site that targets NEMO to the plasma membrane: NEMO-NM) [38] or WT NEMO (Fig. 4c). Only CA-IKK2 and NEMO-NM (but neither WT IKK2 nor WT NEMO) inhibited the cytotoxic effects of the Flt3 knockdown (Fig. 4d) and that of pharmacological Flt3 inhibition (Fig. 4e). Similar results were obtained in MOLM-13 MDS
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Fig. 5 Direct impact of Flt3 inhibition on IKK2. (a) Co-immunoprecipitation of Flt3 with IKK2. Untreated KG-1, MOLM-13 or MV4-11 cells were lysed and subjected to immunoprecipitation with an antibody specific for Flt3, followed by immunoblot detection of IKK1 and IKK2. (b) Effect of Flt3I on the interaction between Flt3 and IKK2. MOLM-13 or MV4-11 cells were left untreated or were treated with Flt3I (2 lM, 2 h), followed by immunoprecipitation of Flt3 and detection of IKK2. (c, d). Flt3I inhibits IKK2 phosphorylation on tyrosine residues by Flt3 kinase. By fluorescent kinase assay, increasing doses of Flt3I inhibited phosphorylation of IKK2 by Flt3 kinase. Immunoprecipitation revealed tyrosine of IKK2 in both MOLM-13 and MV4-11 cell lines which was inhibited by Flt3I
cells (Fig. 4) and MV4-11 AML cells (not shown), in line with the hypothesis that Flt3 depletion/inhibition kills through an inhibitory effect on the IKK complex. Moreover, these results provide unequivocal evidence that Flt3 depletion/inhibition kills the cells through a specific pathway (that involves the IKK/NF-jB pathway) rather than through non-specific effects. Third, we investigated whether Flt3 might engage in direct interactions with the IKK complex. Following immunoprecipitation of Flt3 in MOLM-13 and MV4-11
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cells, we detected IKK2 (but not IKK1) in the precipitate, suggesting that both kinases might interact within the same polyprotein complex (Fig. 5a). This interaction was strongly diminished upon addition of Flt3I to the cells, as seen by a reduction in the amount of IKK2 that immunoprecipitated with Flt3 (Fig. 5b). When IKK2 was immunoprecipitated, NEMO appeared to belong to the complex but no IKK1 was detected (data not shown). As a further indication for a direct regulatory interaction between Flt3 and IKK2, we found that recombinant Flt3I was able to phosphorylate an IKK2-derived peptide in vitro (Fig. 5c), demonstrating that IKK2 can be a direct substrate of Flt3. Indeed, we found that IKK2 is constitutively tyrosine-phosphorylated in MOLM-13 and MV4-11 cells. This phosphorylation was lost after treatment of the cells with Flt3I (Fig. 5d). Altogether, these results indicate that Flt3 inhibition has a direct impact on the subcellular localization and the phosphorylation status of IKK2 in MOLM-13 and MV4-11 cells, correlating with inhibition of the NFjB system. Ex vivo therapeutic effects of Flt3 inhibition on primary MDS and AML cells. The data presented here have been obtained on cell lines and hence must be interpreted with caution as to their possible clinical relevance. We therefore studied the impact of Flt3I on primary, purified CD34+ bone marrow mononuclear cells (BM-MNC) from a cohort of MDS and AML patients (Table 1). However, the vast majority of high-risk MDS and AML patients showed CD34+ BM-MNC stained positively for phospho-Flt3, correlating with constitutively active NFjB (Table 1). None of the MDS and AML patients included in this study exhibited the internal tandem mutation of Flt3 nor FLT3 Asp(835) (Table 1). CD34+ cells from normal donors or patients were cultured either alone or in the presence of Flt3I for 18 h ex vivo, followed by assessment of their spontaneous and Flt3I-induced mortality, as summarized in Table 1 and depicted in Fig. 6a, b. Flt3I had no significant cytotoxic effects on CD34+ bone marrow cells from five distinct healthy donors (Fig. 6b). As reported [17, 39–42], CD34+ BM cells from low-risk MDS patients succumbed to apoptosis spontaneously, and this enhanced baseline mortality was not accelerated by Flt3I (Fig. 6b). In contrast, CD34+ bone marrow cells from high-risk MDS patients and AML patients exhibited a significant (P \ 0.01, paired Student t-test) apoptotic response to Flt3I (Fig. 6a, b). Next, we assessed parameters of IKK and NFjB inhibition in several AML and high-risk MDS patients whose CD34+ bone marrow cells responded to Flt3I ex vivo (Fig. 6c). For both AML and high-risk MDS patients, we found that Flt3 inhibition reduced the constitutive activation of NFjB, as indicated by a loss of the nuclear p65 immunofluorescence staining (Fig. 6d, e; Table 1).
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Fig. 6 Effect of Flt3 inhibition on constitutive NFjB ativation and cell death of purified CD34+ bone marrow cells from MDS and AML patients. (a) Representative DiOC6(3)/PI staining of CD34+ AML bone marrow cells cultured for 18 h in the absence or presence of Flt3I and/or Z-VAD-fmk (as in Fig. 1b). (b) Spontaneous and Flt3I induced cell death in cells from healthy normal volunteers, low-risk MDS, high-risk MDS, and AML (same patients as Table 1). (c) Effect of Flt3I on the presence of the p65 NFjB subunit of CD34+ AML bone marrow cells (same patient as in A), as determined by immunofluorescence. (d) Inhibition of NFjB activation in a cohort of patients with determined blast counts. Lines link individual patients values before and after addition of Flt3I. (e) Nuclear NF-kB in the absence or in the presence of Flt3I for the patients listed in Table 1. Statistical analyses (*\0.05; **\0.01) were performed by means of the paired Student t-test
Altogether, these data allow us to draw two major conclusions. First, constitutive NFjB activation in high-risk MDS and AML correlated with constitutive Flt3 activation (as judged from its phosphorylation status). Second, inhibition of Flt3 reduces this malignancy-associated NFjB activation, presumably through the same pathway that has been delineated for MOLM-13 and MV4-11 cell lines.
Discussion The results presented in this article indicate that Flt3 may be (one of) the upstream kinase(s) that account for constitutive NFjB activation in high-risk MDS and AML. This notion is based on the comparative analysis of Flt3 inhibition (either by means of a pharmacological inhibitor that lacks an off-target
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effect on IKK2, Flt3I, or using distinct small-interfering RNAs) in MDS and AML cell lines or patient-derived, primary bone marrow cells. The epistatic and functional analyses, as well as the correlative data obtained on clinical samples, provide a coherent vision of the hierarchy between IKK and Flt3. Flt3 appears to be constitutively active and hence manifests an activating (auto)-phosporylation on tyrosines 589 and 591, in particular in high-risk (but less so in low-risk) MDS and AML cells. When active, Flt3 associates with IKK2 (but not or only loosely with IKK1). This step most probably occurs close to or at the plasma membrane and involves tyrosine phosphorylation of IKK2 by Flt3. It leads to activation of the IKK complex resulting in NFjB activation (through the phosphorylation and degradation of IjB). Inhibition of Flt3 strongly reduced the tyrosine phorphorylation of IKK2, dissociates the Flt3/IKK2 complex, thereby preventing NFjB activation and triggering
Apoptosis (2008) 13:1148–1161
apoptosis in those cells that are ‘‘addicted’’ to NFjB. As a caveat, it has to noted however, that the effect of Fl3I on NFjB activation and apoptosis induction do not correlate completely in clinical samples and that myeloblasts from some high-risk MDS patients did not manifest a major increment of apoptosis upon culture with Flt3I, pointing to a poorly understood heterogeneity in the patient population. Thus, a majority of AML and high-risk MDS patients follow the pattern delineated above. Nonetheless, a sizeable minority may demonstrate apoptotic responses to Flt3 inhibition without that NFjB would be involved in this process and/or activate NFjB through Flt3-independent pathways. Although these results move us one step further towards the comprehension of constitutive NFjB activation in high-risk MDS and AML, they lead us immediately to the next, yet unresolved question. If an activated Flt3 accounts for IKK activation, what are the molecular mechanisms involved in Flt3 activation? The MOLM-13 and MV4-11 cell lines have been shown to possess internal tandem duplication that activate the tyrosine kinase activity of Flt3, rendering it independent from the ligand [43]. However, activating Flt3 mutations have only been found in a minority of MDS (*5%) and AML (25%) patients [2, 11]. Nevertheless, in our cohort, most high-risk MDS and AML patients expressing phosphorylated Flt3 responded to Flt3I ex vivo, suggesting that Flt3 activation may occur independently from activating mutations in the coding region of the gene [44]. A more detailed molecular analysis of possible Flt3 gene amplifications [3], autocrine feed forward loops [4], promoter hyperactivation of Flt3 or yet-to-bediscovered upstream activators will be required to unmask the molecular etiology of Flt3 activation in these hematopoietic malignancies. Although the initial observations that AML and highrisk MDS cell survival depends on the constitutive activation of IKK suggest the clinical use of IKK inhibitors, whole-body inhibition of the classical NFjB activation pathway could have major side effects, due to the expected major toxic effects of global NFjB inhibition [45]. Indeed, agents like bortezomib that have initially been viewed as ‘‘NFjB inhibitors’’ likewise exert their anti-neoplastic effects through unrelated mechanisms, for instance by the transcriptional induction of the pro-apoptotic protein Noxa [46]. The discovery that IKK is subordinated to Flt3, at least in a fraction of AML and high-risk MDS patients, suggests the tantalizing perspective of inhibiting a kinase with a cell type-specific expression profile. Moreover, as the mouse knockout of Flt3 is viable [47], it can be expected that therapeutic Flt3 antagonists might present acceptable levels of toxicity [21, 48, 49]. The results presented here support this general idea and unravel the mechanism through which Flt3 inhibition kills malignant myeloblasts in AML and high-risk MDS.
1159 Acknowledgements We are indebted to Jalil Abdelali (Institut Gustave Roussy, Villejuif, France) for support in confocal microscopy. Guido Kroemer is supported by Agence Nationale de Recherche, Fondation de France, Cent pour Sang la Vie, Cance´ropoˆle Ile-deFrance, Institut National du Cancer, Ligue Nationale contre le Cancer, and European Community (Active p53, Apo-Sys, TransDeath, RIGHT, ChemoRes, ApopTrain). Jennifer Grosjean received a post-doctoral fellowship by Cance´ropoˆle Ile-de-France. Lionel Ades receives a scholarship from Assistance Publique-Hopitaux de Paris and Caisse Nationale d’Assurance Maladie des Professions Inde´pendantes. M.T. receives a Ph.D. fellowship from Universite´ Paris Sud, Paris 11.
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