Blockade of Integrin-?4- Mediated Adhesion of T-ALL Cells
- 1. Department of Pediatrics, University of Southern California Keck School of Medicine, USA
- 2. German Red Cross Blood Service and Institute for Transfusion Medicine and Immunohematology, Goethe University, Germany
Abstract
Objective: We showed previously that α4 blockade using a humanized monoclonal antibody against integrin α4, Natalizumab, antagonizes stromal adhesion of pre-B ALL cells and in combination with chemotherapy eliminates resistant pre-B-ALL in an MRD setting. Here we determined the effect of Natalizumab on adherence of patient-derived T-ALL cells in vitro and on survival prolongation of murine recipients of T-ALL cells, to explore the potential of Natalizumab as a novel T-ALL treatment strategy.
Methods: Adhesion to stromal matrix proteins in the presence/absence of Natalizumab and survival of murine recipients of primary T-ALL cells treated with/ without Natalizumab were assessed, using previously described methods.
Results: Natalizumab inhibited adhesion of patient-derived T-ALL cells and attenuated leukemia progression, resulting in prolonged survival of recipient NOD/ SCID IL2R gamma-/- mice of patient-derived T-ALL.
Conclusion: α4 blockade interferes with adhesion of T-ALL cells to its counter receptor and thus merits evaluation as a novel adjuvant strategy for T-ALL. Further study is needed to explore at the molecular level the effect of α4 blockade in T-ALL.
Citation
Hsieh YT, Shishido SN, Osborne A, Garvey C, Gang EJ, et al. (2014) Blockade of Integrin-α4-Mediated Adhesion of T-ALL Cells. Ann Pediatr Child Health 2(2): 1012.
Keywords
• T-ALL
• Xenograft model
• Drug resistance
• Integrin α4
• Natalizumab
ABBREVIATIONS
BM: Bone Marrow; IP: Intraperitoneal; MST: Median Survival Time; NZM: Natalizumab; Ig: Immunoglobulin; FD: Found Dead; hCD2: Human CD2.
INTRODUCTION
Leukemia relapse still occurs despite the improvement in overall prognosis and survival of patients with acute lymphoblastic leukemia (ALL). ALL cell survival during exposure to chemotherapy is contributed in part to the cell-cell contact of bone marrow stromal and leukemia cells [1-3]. The integrin heterodimer VLA4 (Very Late Antigen-4) is composed of the integrin α4 (α4) chain in association with the integrin ß1 chain [4,5], which is capable of binding the counter receptors fibronectin, osteopontin (OPN), or VCAM-1[4-6]. VLA4 has been shown to regulate both homing and adhesion, as well as the engraftment of hematopoietic progenitors [7] and ALL cells [8] in BM. In acute myeloid leukemia (AML) cells VLA4 was shown to be a dominant adhesion molecule [9], suggesting that the expression of α4 may be an unfavorable risk factor in AML. It is important to note that alternative observations have been reported [10].We previously showed that α4 is a central molecule for chemoprotection of pre-B ALL and that α4 blockade can sensitize pre-B ALL to chemotherapy [11]. It has been shown that BM stromal cells contribute also to the survival of T-ALL cells, partially through adhesion signaling systems [12-14]. However, formal studies of the potential therapeutic effect of targeting α4 in T-ALL have not been performed. As T-cell-ALL occurs in 10%– 15% of pediatric and in 25% of adult ALL cases [15,16], and since drug resistance remains as much of a problem as in pre-B ALL [17], novel therapies against T-ALL are needed. Here, we evaluate the effect of interference of α4-mediated adhesion of T-ALL cells using the humanized monoclonal anti-α4 antibody Natalizumab.
MATERIALS AND METHODS
Engraftment of primary T-ALL in a xenograft model
Bone marrow samples from T-ALL patients were obtained (LAX1R, SFO5, SFO6) in compliance with the Institutional Review Board regulations of each institution. Informed consent was obtained from all human subjects. Under IACUC approved protocols, NOD/SCID/IL2Rγ-/- (NSG) mice of 5-10 weeks of age were conditioned with a single dose of 250 cGy of total body irradiation, followed by tail vein injection of fresh patient cells as described previously [11,18]. Animal care was in accordance with institutional guidelines
Flow cytometry
Antibodies FITC human CD2 (G46-6) and PE human CD49d (9F10) as well as respective isotype controls were purchased from BD Biosciences.
CD2+ T cells
Mononuclear cells of peripheral blood from healthy donors were isolated by Ficoll and sorted by BD FACSAria II cell sorter (BD Biosciences).
Adhesion assay
T-ALL (LAX1R) cells were either pre-treated with Natalizumab (NZM) or corresponding control IgG4 (G17-4) (BD Biosciences), for 30 minutes and washed once with PBS. Cells were then loaded in triplicate onto 12-well plates coated with 10mg/ml human recombinant VCAM-1 (R&D Systems, Minneapolis, MN) [19]. After 2 hours of incubation, suspension cells in the supernatant were removed and the plate was washed once with PBS. The adhering cells were photographed by Olympus IX71 microscope with 100X magnification and then detached by pipetting 20 times. The cell count for adherent cells was assessed by trypan blue exclusion of dead cells.
Integrin α4 blockade by Natalizumab in vivo
LAX1R cells were lentivirally labeled with firefly luciferase as previously described [11] and injected into sub lethally irradiated NOD/SCID/IL2Rγ-/- mice (5x104 cells/mouse). Control groups received control Ig. Natalizumab was given intraperitoneally (i.p.) (5mg/kg of mouse total body weight once per week) for 4 weeks. Leukemia progression was monitored by bioluminescent imaging.
RESULTS
Integrin α4 is highly expressed in primary T-ALL
Analysis of α4 expression on three cases of patient-derived T-ALL cells by flow cytometry indicated high expression in all three T-ALL samples (Figure 1, left panel),similar to CD2+ cells of healthy donors (Figure 1, right panel).
Integrin α4 blockade inhibits adhesion of primary T-ALL
We previously showed that α4 blockade using Natalizumab can inhibit adhesion of pre-B ALL cells to VCAM-1[13]. To determine the effects of integrin α4 antagonism on cellular adhesion of T-ALL cells, LAX1R cells were incubated withα4 blocking antibody, Natalizumab (NZM). Matched isotype antibody was used as control. NZM treatment led to a significant reduction in the number of VCAM-1 adherent cells when compared with control IgG4 exposure (3.67±1.2% vs. 72±4%; p<0.05) (Figure 2A and 2B).
Targeting Integrin α4 delays the progression of primary T-ALL cells in vivo
Luciferase-labeled LAX1R cells were injected into NOD/SCID/ IL2Rγ-/- mice and subjected to 4 weekly injections of NZM (5mg/ kg/mouse/day) or control Ig from Day 3 post-leukemia injection. Whole-body in vivo bioluminescent imaging 18 days after cell injection showed a marked decrease in T-ALL progression (Figure 3A). Overall, the integrin α4 blockade significantly prolonged survival of T-ALL recipient mice compared to control Ig-treated mice (MST=28 days vs. MST=17.5days; p=0.008; Figure 3B).
DISCUSSION
Integrins engage with cell surface ligands and extracellular matrix (ECM) components, such as fibronectin, collagen, and laminin. Outside the hematopoietic system they bear significant roles in embryogenesis, growth and repair, and haemostasis [20]. On blood cells, they are involved in a diverse number of leukocyte adhesion-dependent functions, with critical roles in inflammation and immune response and stem cell retention [21,22]. High α4 integrin expression has previously been reported for preB-ALL where it contributes to the survival of ALL cells in the presence of chemotherapy [11]. Antagonism of integrin α4 was proposed as a mobilizing strategy for hematopoietic stem cells [23-26]. We have shown that using humanized anti-α4 antibody, Natalizmab, as a novel de-adhering strategy against pre-B-ALL, leukemia cells were de-adhered from their counterreceptor VCAM-1 and sensitized to chemotherapy [11]. Integrins play also a role in protection of T-ALL cells against cytokine withdrawal, activation-induced cell death [13] and ligation of death receptors in endothelial cells [27,28]. A role of integrins has recently been implicated in poor-prognosis T-ALL patients [29]. Therefore, antagonizing α4-mediated adhesion of T-ALL cells could be used as a novel strategy against T-ALL. So far, the underlying mechanism accounting for fibronectin/β1 integrin-dependent survival involves activation of the PI 3-kinase/AKT pathway or collagen-mediated activation of MAPK/ERK pathway [14,30]. Recently, a novel microtubule targeting compound, PBOX15, has been shown to down-regulate β1-, β2- and α4-integrin expression and to disrupt integrin-mediated adhesion of a relapsed childhood T-ALL cell lines (CCRF-CEM) and a BCRABL positive adult B-ALL cell line (SD-1) [31], highlighting the promise of targeting integrins in T-ALL. We have shown that integrin α4 antagonism using a humanized monoclonal antibody, which is in clinical use against multiple sclerosis, de-adheres leukemia cells and that this monotreatment already leads to prolongation of murine recipients of T-ALL. Similar effects were observed in mice bearing primary pre-B ALL cells and, since in that model the combination with conventional chemotherapy led to leukemia eradication, leads us to propose similar effects of α4- blockade on T-ALL as on pre-B ALL treatment. Whether leukemia cell are mobilized to the periphery, as we have shown in a xenograft model of pre-B ALL treated with Natalizumab [11], and whether Natalizumab leads to apoptosis or otherwise sensitizes to chemotherapy remains to be determined.
CONCLUSION
Taken together, the results presented above demonstrate a role of integrin α4 in T-cell leukemia adhesion in vitro and in vivo. In conjunction with published data on pre-B ALL, these data suggest α4 blockade as a novel paradigm with broad antileukemic specificity, while further formal studies are warranted to evaluate α4 as a target for therapeutic intervention in T-ALL.
ACKNOWLEDGEMENTS
Supported by grants from NIH R01CA172896, Hyundai Hope on Wheels, V-foundation and St.Baldrick’s Foundation (YMK). HBo acknowledges research support from LOEWE Cell and Gene Therapy Frankfurt, funded by Hessian Ministry of Higher Education, Research and the Arts ref.no.: III L 4 518/17.004 (2010/2013).
