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which involved protein palmitoylation and histone acety- lation.11-13 Overall, the effects of 2BP on leukemia and its cellular targets remain obscure.
Acute promyelocytic leukemia (APL) is a M3 subtype of acute myeloid leukemia(AML) genetically characterized by chromosome translocations involving retinoic acid receptor α (RARα) on chromosome 17 and promyelocytic leukemia (PML) on chromosome 15, which generates the oncogenic PML-RARα fusion protein.14-17 RARα is a lig- and-dependent transcription factor that binds as het- erodimers with retinoid X receptor (RXR) to their target response elements and plays pivotal roles in a series of physiological processes including cell growth, differentia- tion, survival, and death. The PML-RARα fusion protein, however, acts as a transcriptional repressor of RARα-tar- get genes and results in maturation arrest of myeloid pro- genitors at the promyelocytic stage.15,18
All-trans retinoic acid (ATRA), a natural ligand for RAR, is the first Food and Drug Administration-approved drug for APL differentiation, which degrades PML-RARα complex through caspases-dependent or proteasome pathway. Combinations of ATRA, arsenic trioxide (ATO) or chemotherapy were developed and have dramatically improved the complete remission (CR) rate and survival time of APL patients.15,19-21 However, current ATRA-based regimens may cause adverse events including fatal retinoic acid syndrome, systemic infection or secondary leukemia.15,22,23 In addition, 5-10% of APL patients fail to respond to the therapy targeting PML-RARα or relapse after CR.24 More recently, resistance to ATO in APL was reported by several groups.24-26 Therefore, it is essential to further optimize ATRA-based therapy for better prognosis of de novo or relapsed APL patients. In the present study, 2BP was identified to present synergistic differentiation induction with ATRA in APL cells and murine model. Moreover, 2BP overcomes ATRA resistance in ATRA-resistant cells and leukemic mice. We expected that 2BP would be a promising candidate for APL therapy, especially for overcoming ATRA resistance of relapsed APL patients.
Table 1. Patient data and response to 2BP and/or ATRA.
Methods
Patients and cells
Bone marrow samples were collected from 11 cases of newly diagnosed APL patients at the Department of Hematology of the Second Hospital of Dalian Medical University. Patients were diag- nosed according to the French-American-British classification. Detailed information of patients is listed in Table 1. Informed con- sent was obtained from all patients in accordance with the Declaration of Helsinki, and all manipulations were approved by the Medical Science Ethic Committee of Dalian Medical University. Mononuclear cells were isolated by density gradient centrifugation using Lymphoprep, and cryopreserved. In addition, 3 potential donors for allogeneic bone marrow transplantation were used to purify normal healthy hematopoietic cells. Human CD34+ cells were enriched from bone marrow mononuclear cells using MiniMACS (Miltenyi Biotech, Bergisch Gladbach, Germany) following the manufacturer’s instructions.27 Confirmation of CD34+ cells’ phenotype and purity was assessed by flow cytometry analysis using CD34-PE-Cy7 (BD Biosciences, San Diego, CA). Purified CD34+ cells were grown in serum-free hematopoietic growth medium (HPGM; Lonza) supplemented with 10 ng/mL recombinant human interleukin-3 (rhIL-3), 10 ng/mL rhIL-6 and 50 ng/mL recombinant human stem cell factor (PeproTech). The primary APL cells, AML cell lines NB4, HL60, NB4-MR2, NB4-LR1, and NB4-LR2 were maintained in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL) in a humidified incubator at 37 °C and 5% CO2/95% air (v/v).
Reagents and antibodies
ATRA, arsenic trioxide, 2-Bromopalmitate(2BP), palmitate acid (PA), 16BP and 12BP, DNase-free RNase A and propidium iodide were obtained from Sigma. Rabbit polyclonal antibodies against RARα, RXRα, Vinculin and PML were obtained from Santa Cruz Biotechnology (Santa Cruz). Rabbit polyclonal antibodies against pyruvate kinase M2(PKM2) and β-actin were obtained from Cell Signaling Technology. Anti-PML-RARα fusion antibody was from Abcam.
2-Bromopalmitate promotes APL differentiation
No. Sex Age(years) Chromosome
Blast WBC(x109/L) Vehicle
CD11b positive cells% ATRA
P
<0.001
<0.01 <0.05 <0.001
<0.05
<0.001
<0.01
>0.05
>0.05
>0.05
>0.05
1 M 26
2 F 33 3 M 40 4 M 43
5 F 43
6 M 53
7 M 60
8 M 17 9 F 26 10 M 63
11 M 67
ND
ND
ND
46,XY,t(15;17)
(q22;q12)
ND
46,XY,-8,+22,t(15;17) (q22;q12) 46,XY,+10,i(11q), -13,-14,t(15;17)(q22;q12) 46,XY,t(15;17)(q22;q12) 46,XX
ND
46,XY,t(15;17)(q22;q12)
60.00% 37.88
58.50% 11.21 92.00% 37.55 69.50% 1.3
89.70% 3.16
77.00% 17.42
87.50% 4.73
89.37% 1.05 88.50% 2.2 92.00% 5.29
56.00% 1.6
2.10±0.72
0.40±0.31 2.80±0.93 1.10±0.17
3.04±0.11
0.25±0.19
19.8±2.22
14.52±2.44 1.40±0.50 8.80±1.33
12.80±1.23
2BP
2.78±0.35
1.80±0.74 3.01±0.89 1.42±0.43
12.8±0.11
5.11±0.38
19.44±2.30
16.22±1.90 1.07±0.92 10.51±1.55
11.85±1.87
31.30±2.12
34.17±0.77 36.56±2.33 38.28±2.90
33.13±1.25
29.51±0.22
61.55±4.13
41.85±1.55
29.75±1.73
46.25±3.01
45.87±0.30
ATRA/2BP
59.11±2.44
61.65±0.93 60.40±0.69 65.02±4.13
57.83±3.02
59.66±1.51
75.18±5.50
48.20±2.13
31.22±1.51
45.11±0.73
40.33±1.33
Mononuclear cells from bone marrow of 11 APL patients were isolated by density gradient centrifugation using Lymphoprep and maintained in RPMI 1640 medium supple- mented with 10% FBS.The cells were treated with 10 mM 2BP and/or 10-7M ATRA for 3 days and CD11b-positive cells were counted by flow cytometry. ND indicates not done.P: P value between ATRA and ATRA/2BP.
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