Professor Yong-Mi Kim, Department of Pediatrics, Division of Hematology-Oncology, Children's Hospital Los Angeles, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, 4650 Sunset Boulevard, Mailstop #57, Los Angeles, CA 90027, USA
Treatment of resistant or recurrent acute lymphoblastic leukemia (ALL) remains a challenge. It was previously demonstrated that the adhesion molecule integrin α4, referred to hereafter as α4, mediates the cell adhesion-mediated drug resistance (CAM-DR) of B-cell ALL by binding to vascular cell adhesion molecule-1 (VCAM-1) on bone marrow stroma. In addition, it was previously observed that the blockade of α4 with natalizumab or inhibition using the small molecule antagonist TBC3486 sensitized relapsed ALL cells to chemotherapy. However, α4-targeted therapy is not clinically available for the treatment of leukemia to date. In the present study, the use of a novel non-peptidic small molecule integrin α4 antagonist, AVA4746, as a potential new approach to combat drug-resistant B-ALL was explored. An
Although rapid progress has been made in the development of treatment strategies for acute lymphoblastic leukemia (ALL) over the past decades, prognosis for patients with relapsed or treatment-refractory ALL remains poor (
In previous study, its was shown that natalizumab (NZM), a humanized anti-integrin α4 monoclonal antibody, can cause the de-adhesion of B-ALL cells from VCAM-1, thus sensitizing ALL cells to chemotherapy (
As described previously (
AVA4746 and TBC3486 were obtained from Aviara Pharmaceuticals, Inc. for research use. MG132 was obtained from Selleck Chemicals. Vincristine, Dexamethasone and L-asparaginase were obtained from Children Hospital of Los Angeles.
After harvesting, B-ALL cells or HUVECs (0.1-1x106) were centrifuged at 500 g for 5 min at room temperature and resuspended in 100
For apoptosis assay, B-ALL cells were treated for 48 h with either DMSO or AVA4746 25
As previously described (
Cell adhesion and de-adhesion assay. For the cell adhesion assay, non-tissue culture 96-well plates were coated with 10
Similarly, B-ALL cells (1x106/well) were seeded into the plates pre-coated with 10
B-ALL cells were treated with AVA4746 (0,1 µM, 5
B-ALL cells were first serum-starved by being washed twice with Dulbecco's PBS (DPBS) and cultured in MEM-α media at 37˚C and 5% CO2 overnight. Following another wash with DPBS, B-ALL cells were treated with either the vehicle control 0.1% DMSO or AVA4746 (5 or 25
Mobilization of leukemia cells was tested according to a previously described protocol (
A Matrigel-coated 48-well plate (#354508, Biocoat™; BD Biosciences) was first pre-warmed at 37˚C for 30 min. HUVECs (4-6x104/well) that were treated with AVA4746 (0, 5 or 25
Primary B-ALL cells (0.05x106) were intravenously injected into each NSG mouse. In total of 91 mice were divided into four treatment groups: PBS (5 mice in LAX7R exp#1, 4 mice in LAX7R exp#2, 7 mice in RS4;11, 5 mice in TXL3), AVA4746 group (5 mice in LAX7R exp#1, 5 mice in LAX7R exp#2, 7 mice in RS4;11, 6 mice in TXL3), VDL group (6 mice in LAX7R exp#1, 5 mice in LAX7R exp#2, 6 mice in RS4;11, 6 mice in TXL3), and VDL+AVA4746 group (6 mice in LAX7R exp#1, 5 mice in LAX7R exp#2, 8 mice in RS4;11, 5 mice in TXL3). AVA4746 (15 or 30 mg/kg dissolved in PBS) was then administered by oral gavage twice a day continuously for 14, 21 or 28 days, with treatment starting 2 days after B-ALL cell injection. Subsequently, starting at 3 days after B-ALL cell injection, vincristine (0.5 mg/kg) was administered (i.p.) once a week for 2 or 4 weeks, whilst dexamethasone (10.5 mg/kg) and L-asparaginase (1500 IU/kg) were administered (i.p.) once a day (but not at weekends) for 2 or 4 weeks. Animals were monitored daily and the survival time of the mice was then recorded. Animals were sacrificed by CO2 euthanasia at an air displacement rate of 30% if >15% weight loss was observed or physical changes, including loss of ambulation, were observed.
Statistical significance was assessed using a two-tailed Student's t-test and the data are presented as the mean ± SD. One-way analysis of variance (ANOVA) with Tukey's post hoc test was used for comparisons of > two groups. Survival data were analyzed by Kaplan-Meier survival curves and comparisons were performed using the log-rank test for two groups without adjustment. P<0.05 was considered to indicate a statistically significant difference. All statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, Inc.). At least two of experimental repeats for
Since AVA4746 is a small molecule mimetic compound of VCAM-1(
Other soluble integrin ligands present in the serum may interfere with VLA-4 ligand binding (
Primary B-ALL cells derived from four human patients (LAX7R, LAX7, LAX53 and LAX56) and the B-ALL cell line REH were used to evaluate the on-target effect of AVA4746 on B-ALL
To investigate the competing ligand effect, the de-adhesion assays were performed as previously described (
To determine the role of integrin α4 in B-ALL cell metastasis, mobilization assays were performed. In brief, AVA4746 treatment was initiated when detecTable B-ALL cells could be observed in PB. Subsequently, mice were sacrificed before the PB, BM and SPC were harvested for analysis. When ~2.5% LAX7R cells was detected in the PB (
After 8 h of treatment, PB, BM and SPCs were harvested for a human leukemic population analysis using flow cytometry. There was an insignificant but decreasing tendency of leukemia (% of hCD45+hCD19+) in the PB collected from the AVA4746 group compared with that in the PBS group (
The timeframe of the initial treatment was subsequently optimized, so that when the leukemia cell (hCD45+hCD19+) percentage in the PB was 0.5%, the duration of AVA4746 treatment was prolonged to 36 h (
Results from the
To further determine the effects of AVA4746 on the survival of human B-ALL cell mouse xenograft models, LAX7R cells (0.05x106 cells per mouse) were injected into the NSG mice intravenously. Subsequently, 2 days post-injection, either AVA4746 or PBS was administered by oral gavage at 15 mg/kg, twice a day (30 mg/kg/day) for 14 days. VDL was given 3 days post-injection for 4 weeks by i.p. (
In addition, mouse xenograft models were established using cell lines derived from two other cases B-ALL (RS4;11 and TXL3). AVA4746 in combination with VDL (n=8; MST, 105 days) did not lead to a statistically significant improvement in the survival of mice injected with RS4;11 cells in the VDL-alone group (n=6; MST, 95 days;
Integrin α4 has long been of interest in the hematology research field because it has been frequently reported to be a key mediator of adhesion by leukemia cells to the chemoprotective niche (
The epitopes that can be targeted by the HUTS-21 antibody, termed ligand-induced binding site epitopes, becomes exposed as a result of the conformational change that occur following activation, which can be exploited to discover novel allosteric VLA-4 antagonists (
Subsequently, it was demonstrated that AVA4746 can specifically target human integrin α4 on the cell surface whilst also downregulating the protein expression level of α4 in general. Previous studies have demonstrated that integrins are predominantly degraded through the endosome-lysosomal pathway (
In addition, it was observed that AVA4746 treatment increased the percentage of CD45+/CD19+ leukemia cells in the BM, whilst the percentage of CD45+/CD19+ leukemia cells was decreased in the PB. In endothelial cells, VCAM-1 clustering, either by antibody crosslinking or integrin binding, triggers the activation of Rac1, a Rho-like GTPase (
According to a previously established
Taken together, AVA4746 is a potent antagonist of VLA-4 that can efficiently induce the detachment of B-ALL cells from VCAM-1. It is possible that AVA4746 can exert effects on normal hematopoietic stem and progenitor cells in the bone marrow, since these progenitor cells also express integrin α4(
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
DB, ASW and YMK were responsible for the conceptualization of the present study. YR, EJG, MY and YMK designed the present study. YR, EJG, HNK, SL and HAO performed the research, analyzed and interpreted the data. HCL and MY analyzed the data. All the authors reviewed the draft manuscript, edited the final draft and approved the final version for submission. YMK, YR, EJG, HNK, SL and HAO confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
Bone marrow and peripheral blood samples from patients with B lineage acute lymphoblastic leukemia were acquired in compliance with the Institutional Review Board of Children's Hospital Los Angeles regulations. The human studies were conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from the patients under the approved IRB protocol (approval no. CCI-08-00102). All studies were approved by the Institutional Animal Care and Use Committee of Children's Hospital Los Angeles (Los Angeles, USA).
Not applicable.
The authors declare that they have no competing interests.
AVA4746 has high affinity for VLA4 and efficiently competes with VCAM-1 on B-ALL cells. (A-D) B-ALL cells were stained with PE-HUTS-21 antibody in the presence or absence of 5 mM Mn2+ in HEPES buffer at 37˚C for 30 min. VLA-4 receptor occupancy was plotted against the AVA4746 or VCAM-1 concentration using the sigmoidal dose-response equation with variable slope in the GraphPad Prism software. (A) LAX7R and (B) TXL3 cells were first treated with different concentrations of AVA4746 prior to antibody staining. (C) LAX7R and (D) TXL3 cells were first treated with different concentrations of VCAM-1 prior to antibody staining. Dash curves with blank circles represent the basal level, whereas the solid curve with solid circles refers to the 5 mM Mn2+-stimulated condition. EC50 values and their ranges are shown in each panel. Following 1 h incubation with different concentrations of AVA4746, (E) LAX7R and (F) TXL3 cells were seeded into a 96-well plates pre-coated with human VCAM-1 (10
AVA4746 decreases cell surface and total integrin α4 expression. MFI representing the expression levels of integrin α4 on the (A) LAX7R, (B) LAX7, (C) LAX53, (D) LAX56 and (E) REH cell surfaces 24 and 96 h after treatment with 1, 5 and 25 µM AVA4746, with 0 representing the DMSO control. *P<0.05 vs. 0. Experiments were performed in triplicates. (F) B-ALL cell lines LAX7R, LAX7, LAX53, LAX56 and REH were treated for 24 and 96 h with 1, 5 and 25 µM AVA4746, with 0 representing the DMSO control. Following 24 and 96 h treatment with AVA4746, the integrin α4 protein levels at 140 kDa in LAX7R, LAX7, LAX53, LAX56 and REH cells were analyzed by western blotting, with β-actin being used as lthe oading control. MFI, mean fluorescence intensity; α4, α4 integrin.
AVA4746 causes the detachment of B-ALL cells from human VCAM-1. Primary B-ALL cell lines (A) LAX7R, (B) LAX53, (C) LAX56, (D) TXL3 and (E) ICN24, in addition to B-ALL cell lines (F) RS4;11, (G) SupB15, (H) KASUMI-2, (I) 697, (J) BEL-1, (K) BV173 and (L) RCH were seeded into 96-well plates coated with either human VCAM-1 (10 µg/ml) or 2% BSA as a control for 4 h prior to treatment with the DMSO control or AVA4746 (25 µM) overnight. The percentage (%) of viable adherent cells was calculated based on the cell counts using trypan blue exclusion which was then normalized to those in the VCAM-1-DMSO group. ****P<0.0001. Experiments were performed in triplicates. VCAM-1, vascular cell adhesion molecule-1; B-ALL, B-lineage acute lymphoblastic leukemia.
AVA4746 decreases the number of human B-ALL cells in the mouse peripheral blood samples whilst increasing the number of ALL cells in the bone marrow. (A) Experimental design of the
AVA4746 attenuates tube formation by HUVECs. HUVECs treated with either 0, 5 or 25 µM AVA4746 for 30 min were seeded into Matrigel-coated plates for 4-6 h. (A) Representative flow cytometry dot plot of human integrin α4 and β1 expression in HUVECs before seeding into the Matriel wells. (B) Representative images of the HUVECs are shown. (C) Tube formation was assessed by analyzing the number of nodes (pixels with ≥ three neighboring elements corresponding to a bifurcation), segments (elements delimited by two junctions), meshes (areas enclosed by segments or master segments and made by tube-like structures) and total area, before being subsequently quantified. Images were analyzed with using the ImageJ software and the ‘angiogenesis analyzer’ plugin. *P<0.05, **P<0.01 and ***P<0.001. HUVEC, Human umbilical vein endothelial cells; α4, α4 intergrin; β1, β1 integrin; Nb, number.
Combined AVA4746 and chemotherapy treatment prolongs the survival of mice with primary relapsed B-lineage acute lymphoblastic leukemia. (A) NSG mice were intravenously injected with 50,000 LAX7R cells per mouse and then treated with either AVA4746 or the PBS vehicle control (15 mg/kg by oral gavage twice a day continuously for 14 days) 2 days after injection with LAX7R cells. In addition, 3 days after LAX7R cell injection, mice were treated with VDL alone (0.5 mg/kg vincristine once a week, 10.5 mg/kg dexamethasone five times a week and 1500 IU/kg L-asparaginase five times a week for 4 weeks) intraperitoneally or in combination with AVA4746 by oral gavage. (B) Kaplan-Meier survival analysis for the experimental design shown in (A). The MST was 38 days for both the PBS group (n=5) and the AVA4746 group (n=5). The 68-day MST of the VDL-treated group (n=6) was significantly different from the 78.5-day MST of the AVA4746- and VDL-treated group (n=6). *P=0.01. (C) The same protocol as that performed in (A) was conducted, except for that the dose of AVA4746 was higher (30 mg/kg) and the treatment time was longer (twice a day for 28 days). (D) Kaplan-Meier survival analysis for the experimental design shown in (C). The MST was 46.5 days for the PBS group (n=4), whilst the MST was 46 days for the AVA4746 group (n=5). The 77-day MST of the VDL-treated group (n=5) was significantly different compared with the 87-day MST in the group treated with the combination of both AVA4746 and VDL (n=5). *P=0.023. NSG, NOD.Cg-