Preclinical Evaluation of Adjuvant Therapy with AMD3100 for Drug Resistant Philadelphia Chromosome Positive and Negative ALL

Abstract

Despite advances in chemotherapeutic treatment of acute lymphoblastic leukemia (ALL), 20% of children relapse with high death rates, and adults show a long-term survival of only 60%, so new treatment modalities are needed. AMD3100 is being tested in clinical trails for use as a mobilizing agent for hematopoieitic stem cells and blocks the interaction of CXCL12 (SDF-1a) and its receptor, CXCR4, which are involved in retaining hematopoietic cells in primary lymphoid organs. CXCR4 expressed on bone marrow fibroblasts is involved in retaining pre-B cells in the bone marrow. Blocking the CXCL12 – CXCR4 interaction makes murine acute lymphoblastic leukemia cells more sensitive to drug treatment in vitro. Therefore, we hypothesized, that AMD3100 may sensitize drug resistant leukemia cells to chemotherapy. To test this hypothesis, we established a pre-clinical xenograft model of B-cell precursor-ALL, allowing us to monitor progression of leukemia in vivo with non-invasive bioimaging. Frozen samples of drug resistant adult patients, who did not respond to chemotherapy (drug-resistant Philadelphia chromosome (Ph) positive (Ph+) or negative (Ph−) pre-B ALL leukemia cells), were engrafted via tail-vein injections into female NOD/SCID or NOD/SCID IL2Rγ−/− mice. Primary passages were serially expanded up to three passages. Analysis of the phenotype by flow cytometry and morphology by histochemistry of the xenografts confirmed that the initial characteristics of the patient were retained or not altered throughout the passages. In an effort to sensitize those drug resistant leukemia cells to chemotherapy, we have next proceeded to test AMD3100 in our established preclinical models. Primary ALL cells of an Imatinib-resistant Ph+ patient were transduced with luciferase and injected into sublethally irradiated NOD/SCIDIL2Rγ −/− mice (0.7×106 cells/recipient). Mice were treated per os with saline (n=3), Imatinib (n=4;75 mg/kg/d), AMD3100 via an osmotic pump (n=4;10 mg/kg/d) or Imatinib plus AMD3100 (n=8) for 28 days, after detection of engraftment by bioluminescent imaging. Survival of the group treated with Imatinib + AMD3100 (Median survival time, MST=49 days) was significantly prolonged compared to the group treated with Imatinib only (MST=38 days) (p<0.05). Next, primary ALL cells of a Ph− patient were transduced with luciferase and injected into NOD/SCID mice (0.4×106 cells/animal). Upon detection of engraftment by bioimaging, xenografted mice were treated with saline (n=2), vincristine-dexamethasone-L-asparaginase (VDL; n=3), AMD3100 (n=3) or AMD3100 plus VDL (n=6). Again, significantly prolonged survival of the group treated with the combination of VDL + AMD3100 (MST=61.5 days) compared to the VDL only treated group (MST=54 days) (p<0.05) was observed. In summary, there was a clear beneficial effect of the combination of AMD3100 with two different chemotherapeutic regimens resulting in a significant improvement of survival. Further preclinical evaluation of AMD3100 as a novel adjuvant has the potential to lead to translation into clinical trials.