Targeting of Integrin α4/VCAM-1 with AVA4746 Modulates the Redox-Status and Metabolism in ALL and Angiogenesis

Background. Treatment of resistant or relapsed acute lymphoblastic leukemia (ALL) remains a challenge. Adhesion of ALL cells via integrin α4/VCAM-1 pathway to bone marrow stromal cells promotes cell adhesion-mediated drug resistance (CAM-DR). Previously, we have shown that AVA4746, a non-peptidic small molecule integrin α4 antagonist, functionally de-adheres patient-derived B-ALL cells from VCAM-1. Furthermore, we demonstrated that antagonizing VCAM-1 by AVA4746 in combination with traditional chemotherapy prolongs survival of B-ALL xenograft NSG mice derived from a relapsed B-ALL patient. The objective of the present study was to determine the underlying mechanism of the integrin α4/VCAM-1 pathway blockade using AVA4746.

Method. The effect of AVA4746 intracellular levels of reactive oxygen species (ROS) in five primary B-ALL cells by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFDA) staining. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurement was assessed by Seahorse XF cell mito stress test in one primary ALL using Seahorse XFp Analyzer. The effect of AVA4746 on angiogenesis was analyzed by using an endothelial tube formation assay with HUVEC cells. In vivo effect of high dose of AVA4746 (30mg/kg bid p.o.) and prolonged treatment (28 days) was assessed in a murine xenograft model of primary ALL.

Results. We have previously determined that the metabolic activity was markedly decreased in two of two ALL cases by AVA4746 as assessed by WST-1 assay. Therefore, we investigated further the effect of AVA4746 on metabolism of ALL cells. For this purpose, ALL cells were treated in vitro with AVA4746 (25μM) or 0.1% DMSO as control for 2 or 4 hours. A significant increase of intracellular ROS level was consistently found in presence of AVA4746 compared to DMSO at both 2h or 4hours in all five primary B-ALL cells. According to our preliminary bioenergetics assay, after 24h treatment with AVA4746 0, 25μM, AVA4746 decreased the metabolic potential of LAX7R cells as shown by the difference in stressed OCR and stressed ECAR (P=0.0036 and 0.0056, respectively). AVA4746 significantly impeded endothelial tube formation of HUVEC cells as shown by the statistically significant differences in numbers of nodes, segments, meshes and meshes area indicating an effect of AVA4746 on angiogenesis, which will be further examined in vivo. In vivo, AVA4746 (30mg/kg bid p.o.) in combination with vincristine, dexamethasone, L-asparaginase (VDL) chemotherapy treatment prolonged survival (n=5) compared with the VDL only treated group (n=5) (MST= 87 days vs MST= 77 days; P=0.0229). There was no significant difference in survival between the PBS control group (n=4) and the AVA4746 only treatment group (n=5).

Conclusion. Interrupting integrin α4/VCAM-1 pathway by AVA4746 increases intracellular ROS level of B-ALL cells and downregulates metabolic potential. AVA4746 inhibits in vitro angiogenesis. The combined treatment of high dose AVA4746 and VDL prolonged survival compared to VDL alone. As the integrin α4/VCAM-1 pathway plays a critical role in CAM-DR of B-ALL, AVA4746 may be a potential candidate for therapy of drug resistant B-ALL.