VLA4 Blockade In Acute Myeloid Leukemia

Despite aggressive chemotherapy and early allogeneic transplantation, acute myeloid leukemia (AML) frequently relapses, so that over-all disease-free survival remains below 50%. Strategies to overcome the chemoresistance of relapse-initiating residual AML blasts are, therefore, warranted. Evidence has been provided that AML cells are sheltered from the insult of chemotherapeutic agents by interacting with bone marrow stroma. Integrin alpha4beta1 (VLA4) mediates adhesion of hematopoietic cells to bone marrow stroma cells and extracellular matrix and has been implicated in cell adhesion-mediated chemotherapy resistance. Based on the evidence thereof provided for ALL blasts, VLA4 is here proposed as a therapeutic target for refractory AML. For this purpose, VLA4 was functionally blocked in vitro and in vivo on patient-derived AML cells using an anti-functional humanized VLA4 antibody, Natalizumab (NZM). VLA4-positive (>90%) patient-derived (primary) AML cells were plated on immobilized human VCAM1 or human stromal cell line HS-5 and treated with control (IgG4) or Natalizumab (NZM) for 2 days. NZM de-adhered 94.0%±7.6 AML cells from its counter receptor VCAM-1, yet only 31.3%±13.8 from HS-5, indicating that stroma cells offer ligands for a wider panel of adhesion receptors besides VLA4. We tested also whether VLA4 blockade is beneficial against AML when combined with chemotherapy. For this purpose, primary AML cells were incubated with NZM and incubated on uncoated tissue culture plates or HS-5 stromal layers in the presence or absence of Ara-C (1µM) for two days. AML cells showed higher viability under Ara-C therapy when incubated with HS-5 cells compared to controls, indicating the chemoprotective effect of the stromal layer. The viability of the AML cells treated with combined Ara-C and NZM was similar to the controls, indicating that HS-5-mediated chemo-protection was completely abrogated by NZM. Significantly more AML cells treated with Ara-C+NZM stained AnnexinV+/7AAD- than after Ara-C+control Ig4 treatment (44.4%±5.6 vs. 29.8%±4.8, p=0.03) indicating increased apoptosis of AML cells. On its own, NZM did not induce apoptosis. Next, we tested NZM as a single agent in our NOD/SCIDIL2Rγ deficient (NSG) xenograft model of primary AML. Luciferase-labeled AML cells were intrafemorally injected into NSG mice (1x10⁵ cells / mouse). NZM (5mg/kg) was given intraperitoneally once per week for 4 weeks. NZM-treated animals survived significantly longer than control Ig-treated animals (Median Survival Time, MST=107 days vs. MST=76 days; *p=0.008 by Log-rank Test.To determine effects of NZM on leukemia cell burden/distribution in different organs, primary AML cells were injected into NSG mice and allowed to engraft for 3 days, subsequently treated with a single dose of NZM or Ig control. 72 hours later, AML cell burden in femurs and spleens of NZM-treated animals was significantly decreased compared to control treated mice, however AML cells were not increased into the peripheral blood, so that whether leukemia cells were selectively killed ormobilized and then retained in non-hematopoietic organs remains to be determined. Further studies addressing molecular mechanisms of increased apoptosis after combined VLA4 blockade and chemotherapy are ongoing. Our data suggest that the paradigm of leukemia cell targeting by VLA4 blockade, previously demonstrated by us for ALL, can also be applied to AML.