Adjuvant CD49d Blockade Eradicates Chemoresistant ALL
Authors: Hsieh Yao-Te , Park Eugene , Jiang Enzi , Dauber Katrin , Chudziak Doreen , Schaefer Paul , Klemm Lars , Scharman Carlton , Kang Eun-Suk , Koo Hong-Hoe , Loh Mignon L. , Hofmann Wolf Karsten , Heisterkamp Nora , Muschen Markus , Shimada Hiroyuki , Bonig Halvard , Kim Yong-Mi 
Published: October 2019

Abstract
Abstract 869
Despite the recent advances in chemotherapy for acute lymphoblastic leukemia (ALL), drug resistance resulting in relapse and long-term side effects of current treatments warrant new treatment modalities. Integrin α4β1 (VLA4/ITGA4/CD49d) mediates adhesion of hematopoietic cells onto bone marrow cells and has been implicated in cell adhesion-mediated drug resistance of leukemia cells. Gene expression analyses indicate that VLA4 is upregulated in B-lineage Acute Lymphocytic Leukemia (ALL). Therefore, we hypothesize that VLA4 might be a potential target for treatment of drug resistant ALL To test our hypothesis, we determined the effect of VLA4 inhibition on engraftment of primary pre-B ALLs using a humanized CD49d antibody, Tysabri, as a single agent in our NOD/SCID xenograft model of primary pre-B ALL. Tysabri is known to mobilize normal hematopoietic progenitor cells into the circulation. It blocks binding of VLA-4 to its counter receptors VCAM-1 and osteopontin and we have shown previously in a small pilot study that adjuvant administration with chemotherapy sensitizes one drug resistant primary ALL in vivo to drug treatment. In this study, we injected primary ALL cells from eight different donors into NOD/SCID mice. The samples encompass various cytogenetic aberrations (BCR-ABL, E2A-PBX, MLL-AF, normal karyotype). Cells were luciferase-transduced for in vivo cell tracking and pretreated in vitro with either Tysabri (n=3 per leukemia, n=24 total) or human Ig as a control (n=3 per leukemia, n=24 total). Recipients of Tysabri treated leukemias showed significantly prolonged median survival time (BCR-ABL: MST=112days, E2A-PBX: MST=83days, MLL-AF4: MST=51days; Normal karyotype: MST=48days) compared to control groups (BCR-ABL: MST=84days, E2A-PBX: MST=54days, MLL-AF4: MST=35days; Normal: MST=39days) (p<0.05). Therefore, engraftment of leukemia was significantly delayed in the Tysabri-treated groups as determined by bioluminescent imaging (p<0.05) and survival analysis (p<0.05). Next, we injected two luciferase-labeled pre-B ALLs (US7R, RS4;11) into NOD/SCID mice, which were then treated intraperitoneally with saline (US7R: n=4; RS4;11: n=3), Tysabri (US7R: n=4; RS4;11: n=3), VDL (Vincristine, Dexamethasone and L-Asparaginase) (US7R: n=9; RS4;11: n=5), or VDL+Tysabri (US7R: n=9; RS4;11: n=5), for 4 weeks. Tysabri-treated groups showed prolonged survival time (US7R: MST=52days; RS4;11: MST=83) compared with saline-treated groups (US7R: MST=38days; RS4;11: MST=60 days) (p=0.007). VDL-only treated animals died rapidly (US7R: MST=74days; RS4;11: MST=109 days), however, the animals treated with the combination VDL+ Tysabri, survived disease-free until the end of follow-up (US7R: MST=151days; RS4;11: MST=141 days) (p<0.0001). The sacrificed animals showed absence of human CD45 in spleen, liver, bone marrow and lung by immunohistochemistry and flow cytometry indicating eradication of recalcitrant leukemia cells. We have also shown in vivo using an immunocompetent mouse model that VLA4 ablation does not result in dose-limiting toxicity to normal hematopoietic cells after VDL or 5-FU treatment. To understand further the role of VLA4 deletion in ALL, we established a model of murine leukemia using bone marrow cells from VLA4 floxed mice, retrovirally transformed with BCR-ABL1 p210 and cmyc. Subsequent to leukemic outgrowth, cells were transduced with either Empty GFP control, or Cre-GFP vector to delete VLA4. Knockout of VLA4 in transduced cells was detected by PCR on genomic DNA and by flow cytometry (Empty GFP control: 97% CD49+; Cre-GFP vector: 0.8% CD49+). Upon in vitro culturing of the cells 4-fold more VLA4 deleted cells were found in the supernatant compared to the control cells (p<0.05) determined by Trypan blue exclusion counts of dead cells, indicating that CD49d in murine leukemia is required for cell adhesion. Further functional studies addressing engraftment and gene expression upon induced VLA4 deletion are ongoing. Taken together, our data demonstrate that CD49d-blockade with adjuvant chemotherapy can eradicate chemotherapy-resistant leukemia. Further studies are warranted to understand and evaluate preclinically adjuvant inhibition of integrins to overcome relapse of leukemia.

Disclosures:
No relevant conflicts of interest to declare.