AVA4746, an Orally Available, Clinical Grade Antagonist of Integrin Alpha 4, Sensitizes Pre-B Cell Acute Lymphoblastic Leukemia to Chemotherapy

Background. Even though remarkable progress has been made in the treatment of childhood acute lymphoblastic leukemia (ALL), salvage of relapse patients remains a challenge. The role of the bone marrow (BM) microenvironment is critical to protect leukemia cells from chemotherapy. The BM microenvironment promotes cell adhesion-mediated drug resistance (CAM-DR) in ALL.We and others have shown that the adhesion molecule integrin α4, referred to hereafter as α4, mediates drug resistance of B-ALL. In our previous studies, we showed that both α4 blockade by natalizumab and inhibition by the small molecule α4 antagonist TBC3486 can sensitize relapsed ALL cells to chemotherapy. However, no α4 targeting therapy is currently clinically available to treat leukemia. Here, we preclinically evaluate a novel non-peptidic small molecule antagonist, AVA4746, which has been safely used in clinical studies, as a potential new approach to combat drug resistant ALL.

Method. Six refractory or relapsed primary pre-B ALL cases were used for in vitro studies. Viability was assessed by trypan blue counts or annexin V/7AAD flow cytometric analysis and metabolic activity was evaluated by Cytoscan WST-1 assay. For in vivo evaluation a NOD/SCID IL2Rγ^-/- xenograft model of primary pre-B ALL (LAX7R) was used.AVA4746 (15mg/kg) was administered by oral gavage twice a day continuously for 14 days, and vincristine, dexamethasone, L-asparaginase (VDL) was given intraperitoneally (weekly) for 4 weeks. Overall survival was determined by Kaplan-Meier Survival analysis.

Results. AVA4746 caused a significant decrease in mean fluorescence intensity (MFI) of α4 expression in six out of six ALL cases at doses of both 5μM and 25μM after 24 hours and 96 hours compared to DMSO control. Interestingly, decreased protein expression of α4 was also observed by Western Blot analysis all six ALL cases. We tested next in two cases (LAX53, ICN13), if AVA4746 de-adheres ALL cells from its counter receptor VCAM-1. The percentages of adherence after treatment with AVA4746 (25μM) were significantly lower than after DMSO treatment (10.3%±4.9% vs. 99.9%±7.6%, p= 0.00007 for LAX7R; 8.1%±1.0% vs. 100.1%±13.6%, p= 0.0003 for LAX53; 9.0%±1.6% vs. 100.0%±14.0%, p=0.0004 for ICN13). AVA4746 was not associated with apoptosis in vitro alone or in combination with chemotherapy (VDL). Metabolic activity as assessed by WST-1 assay was markedly decreased by AVA4746 in two of two ALL cases. AVA4746 also decreased ALL proliferation in two out of two ALL samples tested. In vivo, AVA4746 in combination with VDL chemotherapy treatment led to significant prolongation of overall survival (n=6) compared with the VDL only treated group (n=6) (MST= 78.5 days vs MST= 68 days; P<0.05). There was no significant difference in survival between the PBS control group (n=5) and the AVA4746 mono-treatment group (n=5) (MST=38days vs MST= 38days).

Conclusion. We have identified α4 as a central adhesion molecule in CAM-DR of ALL and have shown that AVA-4746, an orally available and specific α4 antagonist, which has been safely used in clinical studies, downregulates α4 in primary ALL and functionally de-adheres them from VCAM-1. Critically, we demonstrated that inhibition of α4 in combination with standard chemotherapy can prolong the survival of NSG mice bearing pre-B ALL. These data support further study of inhibition of α4 using AVA4746 as a novel strategy to treat drug resistant B lineage ALL.