Letters to the Editor
1308
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. doi:10.1038/sj.leu.2404647; published online 15 March 2007
ZD6474 (ZACTIMA) is an orally available, selective inhibitor of tyrosine kinases coding for vascular endothelial growth factor receptor, epidermal growth factor receptor and RET (REarranged during Transfection). ZD6474 inhibits the growth of a broad range of established human tumor xenografts, including those from prostate, breast, ovary and lung.1 In addition, we have recently shown that ZD6474 induced growth arrest and apoptosis of gastrointestinal stromal tumor cells via inhibition of c-KIT signaling.2 This study explored the effect of ZD6474 on a variety of human leukemia cell lines with defined genetic abnormalities. ZD6474 induced growth arrest and apoptosis of EOL-1, MV4-11 and Kasumi-1 cells, which posseses activating mutations in platelet-derived growth factor receptor a (PDGFRa), FMS-like tyrosine kinase 3 (FLT3) and c-KIT, respectively (Table 1). ZD6474 effectively inhibited phosphorylation of PDGFR, FLT3, and c-KIT in these leukemia cells (Figure 1a). In addition, ZD6474 was active against freshly isolated acute myeloid leukemia (AML) cells that had mutations
Table 1 Inhibition of the proliferation of malignant hematopoietic cells by ZD6474 Cell line
Gene alterations
IC50 (mM)a
Eosinophilic leukemia EOL-1
FIP1L1/PDGFRa
0.3
Biphenotypic leukemia MV4-11
FLT3-ITD
0.5
Myeloid leukemia Kasumi-1 U937 THP-1 HL-60 Kcl-22 K562
Bcr/Abl Bcr/Abl
Multiple myeloma U266 Pleural effusion lymphoma KS-1 BCBL-1
Adult T-cell leukemia MT-1 MT-2 MT-4
0.4 Not achieved Not achieved Not achieved 5 5 2.5 2 5
ZD6474 (M)
−
0.1
0.5
1
−
0.1
0.5
1
1.0
0.1
0
p-Tyrosine
A, EOL-1
PDGFR
p-Tyrosine
B, MV4-11
FLT3
p-Tyrosine
C, Kasumi-1
C-KIT
b
ZD6474 (M) p-STAT3
Relative band intensities
1.0
p-AKT Relative band intensities AKT
1.0
0.6
1.0
0.3
0.2
0.1
p-ERK Relative band intensities
0.5
0
ERK
10 6 Not achieved 6 Not achieved Not achieved 10
a Concentration of ZD6474 that induced 50% growth inhibition (IC50) was calculated from dose–response curves.
Leukemia
a
STAT3
c-KIT (Asn822Lys)
Lymphoblastic leukemia PALL-1 Bcr/Abl PALL-2 Bcr/Abl NALL-1
Burkitt lymphoma Daudi
in the FLT3 gene as measured by colony forming assay (Table 2). Additional investigations revealed that ZD6474 blocked the downstream signaling pathways including extracellular signalregulated kinase (ERK) signaling in EOL-1 cells (Figure 1b). Further studies examined the antileukemic activity of AZD6244 (ARRY-142886),3 a novel inhibitor of mitogen-activated protein kinase /ERK signaling. The drug synergistically enhanced the
Figure 1 ZD6474 inhibits autophosphorylation of RTKs and their downstream effectors in leukemia cells. (a), EOL-1, MV4-11 and Kasumi-1 cells were cultured with various concentrations of ZD6474. After 1 h, the cells were harvested and the proteins were extracted. The PDGFRa (EOL-1), FLT3 (MV4-11) and c-KIT (Kasumi-1) proteins were immunoprecipitated and subjected to Western blot analyses. The membrane was probed sequentially with an anti-phosphotyrosine antibody or anti- PDGFRa (EOL-1), anti-FLT3 (MV4-11) or anti-c-KIT (Kasumi-1) antibodies. (b), EOL-1 cells were cultured in the presence of ZD6474 (0.1–1 mM). After 1 h, the cells were harvested and proteins were extracted and subjected to Western blot analysis. The membrane was sequentially probed with anti-phospho-Akt (Ser473), anti-phospho-STAT3 (Tyr705), anti-phospho-ERK (Tyr202/Tyr204), anti-Akt, anti-STAT3 and anti-ERK antibodies, and band intensities were measured using densitometry.
Letters to the Editor
1309 Table 2
Effect of ZD6474 on freshly isolated acute leukemia cells
Pt. No.
Age/Sex
FAB
WBC 109
% Blast
1 2 3 4 5 6 7 8 9 10 11 12 13
80/M 59/M 80/M 32/M 54/F 70/F 51/F 40/F 64/F 74/M 77/F 72/F 68/M
M1 M1 M1 L2 M1 M1 M7 M1 M1 M1 M1 M2 M1
7900 1300 5800 77 100 188 000 27 500 4400 1700 7000 5200 1900 18 900 87 700
56 43 88 54 99 82 63 21 47 86 14 94 96
Genetic abnormalities
FLT3 mutation
5, 7, 8, 13, 14, 16, add(17)(q23) 3, 7, 8, 17, 18, 21, 22, add(18)(p11) t(9;22)(q34;q11), add(8)(q24), add(7)(q34) 7, t(12;15)(p13;q15), add(11)(q23) t(10;11)(p21;q14) del(16)(q?) t(8;21)(q22;q22)
IC50
F F F F ITD F F F F F F ITD ITD
Previous treatment
0.5 not achieved 0.1 0.5 o0.01 0.5 0.8 1 not achieved not achieved 0.9 o0.01 o0.01
No No Yes Yes No No Yes No No Yes No Yes No
The freshly isolated leukemia cells were cultured in methylcellulose medium in the presence of various concentrations of ZD6474 (0.01–1 mM). After 14 days, colonies were counted. The concentration of ZD6474 that induced 50% inhibition of colony formation (IC50) was calculated from the dose– response curves.Pt, patient; M, male; F, female; FAB, French–Amercan–British (leukemia classification); ITD, internal tandem duplication; WBC, white blood cells.
EOL-1 MV4-11
b
c
Cell cycle 75
Antagonism
control ZD6474 0.25M AZD6244 0.25M ZD6474+AZD6244
1.0 50
0.8 Cells (%)
0.6 0.4
25
Synergism
* *
0.2
IC75
pre-G1
IC90
G0/G1
S
G2/M
Effect levels
300
240
M1
180
58%
Counts
* 50
*
25
120
240
M1
240
M1
180
49%
180
44%
120 60
60
0
0
0
102 103 FL1 Height
104
100
AZD6244 (0.25M)
102 103 FL1 Height
104
101
103 102 FL1 Height
M1
240
M1
180
18%
180
5.0%
180
4.8%
Counts
240
120
60
0
0
0
103 102 FL1 Height
104
100
101
103 102 FL1 Height
104
120
300
120
60
35%
100
300
M1
101
104
ZD6474 (0.5M) +AZD6244 (0.25M)
60
M1
180
0 100
240
120
240
60
ZD6474 (0.1M) +AZD6244 (0.25M)
300
300
101
ZD6474 (1M)
300
120
60 101
ZD6474 (M) 0 0.25 0.5 0 0.25 0.5 0 0.25 0.5 AZD6244 (M) 0 0 0 0.25 0.25 0.25 0.5 0.5 0.5
ZD6474 (0.5M)
300
Counts
Counts
ZD6474 (0.1M)
Control 300
Counts
d
100
* *
75
Counts
IC50
Counts
100
0
0.0
100
EOL-1
Counts
Combination index (C.I)
1.2
annexin positive cells (%)
a
101
103 102 FL1 Height
104
ZD6474 (1M) +AZD6244 (0.25M)
240
M1
180
4.4%
120 60 0
100
101
102 103 FL1 Height
104
100
101
102 103 FL1 Height
104
p-ERK p-ERK IgG1
Figure 2
For figure caption see next page.
ability of ZD6474 to induce growth arrest and apoptosis of EOL1 cells in conjunction with further downregulation of p-ERK (Figures 2a-d). Clinical studies showed that the remission in AML patients elicited by sunitinib, an FLT3 kinase inhibitor, was partial and of
short duration,4 suggesting that the addition of other anticancer compounds may be required. Recently, we have shown that blockade of Akt/mTOR signaling by rapamycin synergistically enhanced sunitinib-mediated growth inhibition of leukemia cells.5 Taken together, concomitant administration of the FLT3 Leukemia
Letters to the Editor
1310 kinase inhibitor and downstream signal pathway inhibitor such as rapamycin, as well as AZD6244, may be a potential treatment strategy. In summary, ZD6474 may be an effective treatment for individuals with AML that possess mutations in receptor tyrosine kinase genes. In addition, the combined administration of ZD6474 and AZD6244 to patients with AML warrants further investigation.
Acknowledgements This work was supported in part by Kanae Foundation for the Promotion of the Medical Science, Public Trust of Haraguchi Memorial Cancer Research Fund, AstraZeneca Research Grant 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, T Ikezoe1, A Takeshita1, J Yang1, T Tasaka2, Y Yang1,3, Y Kuwayama1, N Komatsu1, K Togitani1, HP Koeffler4 and H Taguchi1 1 Department of Hematology and Respiratory Medicine, Kochi University, Nankoku, Kochi, Japan; 2 Department of Clinical Pathology and Laboratory Medicine, Kawasaki Medical School, Kurashiki, Okayama, Japan; 3 Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing, China and
4 Department of Hematology and Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA, USA E-mail:
[email protected]
References 1 Wedge SR, Ogilvie DJ, Dukes M, Kendrew J, Chester R, Jackson JA et al. ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration. Cancer Res 2002; 62: 4645–4655. 2 Yang Y, Ikezoe T, Nishioka C, Taguchi T, Koeffler HP, Taguchi H. ZD6474 induces growth arrest and apoptosis of GIST-T1 cells and this growth inhibition is enhanced by concomitant use of sunitinib. Cancer Sci 2006; 97: 1404–1409. 3 Staehler M, Rohrmann K, Haseke N, Stief CG, Siebels M. Targeted agents for the treatment of advanced renal cell carcinoma. Curr Drug Targets 2005; 6: 835–846. 4 Fiedler W, Serve H, Dohner H, Schwittay M, Ottmann OG, O’Farrell AM et al. A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease. Blood 2005; 105: 986–993. 5 Ikezoe T, Nishioka C, Tasaka T, Yang Y, Komatsu N, Togitani K et al. The anti-tumor effects of sunitinib (formerly SU11248) against a variety of human hematological malignancies: enhancement of growth inhibition via inhibition of mTOR signaling. Mol Cancer Ther 2006; 5: 2522–2530.
Figure 2 AZD6244 potentiates the action of ZD6474 in leukemia cells. (a), EOL-1 or MV4-11 cells were cultured with ZD6474 and/or AZD6244 (0.125–1 mM). After 2 days, cell proliferation was measured by thymidine uptake. The percent inhibition was graphed and the concentration of each compound that induced 50, 75 or 90% growth inhibition (IC50, IC75, IC90) was determined (data not shown). The combination index (CI) of ZD6474 and AZD6244 at various dose effects (IC50, IC75, IC90) was calculated using the median effect method. CI values less than 1 indicate synergy, a CI equal to 1 indicates an additive effect, and a CI greater than 1 indicates antagonism between the two agents. (b) Cell cycle analysis. EOL-1 cells were cultured with ZD6474 and/or AZD6244 (0.25 mM). After 2 days, the cell cycle distribution of these cells was analyzed. The statistical significance of difference between populations in pre-G1 or S phase of cell cycle induced by either ZD6474 or AZD6244 alone and those induced by a combination of both was determined by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison tests. Results represent the mean7s.d. of three experiments performed in triplicate; nPo0.01, with respect to either compound alone. (c) Annexin V staining. EOL-1 cells were cultured with ZD6474 and/or AZD6244 (0.25 or 0.5 mM). After 2 days, cells were stained with annexin V/propidium iodide and analyzed by a flow cytometry. The statistical significance of difference between annexin V positive populations induced by either ZD6474 or AZD6244 alone and those induced by combination of both was determined by ANOVA followed by Bonferroni’s multiple comparison tests. Results represent the mean7s.d. of three experiments performed in triplicate; nPo0.01, with respect to either compound alone. (d) FACScan analysis of p-ERK. EOL-1 cells were cultured with either ZD6474 (0.25–1 mM) or control diluent. After 24 h, cells were exposed to either AZD6244 (0.25 mM) or control diluent for 15 min. Cells were harvested, incubated with anti-p-ERK antibody for 30 min at room temperature, and analyzed by flow cytometry. Results represent one of the experiments performed twice in duplicate plate. p-ERK-positive population was quantified using the CellQuest software package. For figure see previous page.
Bevacizumab reduces VEGF expression in patients with relapsed and refractory acute myeloid leukemia without clinical antileukemic activity
Leukemia (2007) 21, 1310–1312. doi:10.1038/sj.leu.2404632; published online 1 March 2007
Angiogenesis is an absolute requirement for tumor growth and metastasis, and is an attractive target for biologically based cancer therapies. Vascular endothelial growth factor (VEGF, VEGF-A) is one of the most potent promoters of angiogenesis whose functions are mainly mediated through the tyrosine kinase receptor VEGFR-2 (also known as KDR or Flk-1). Comparable to solid tumors, there is growing evidence that the VEGF/VEGFR2 signaling pathway plays an important role Leukemia
in leukemic cell growth.1–4 Clinical trials with VEGF inhibition in a variety of malignancies are ongoing. The best-studied and most advanced approach to VEGF inhibition is the recombinant humanized monoclonal antibody bevacizumab (BV). We have treated patients with relapsed or refractory acute myeloid leukemia (AML), not qualifying for further intensive cytotoxic chemotherapy, in order to investigate single agent BV in AML. Nine patients were treated between May 2005 and May 2006. All patients signed informed consent before enrollment. A summary of baseline patient characteristics is shown in Table 1. Patients were between 46 and 81 years old (median:
