Abstract
Adhesion within the bone marrow (BM) microenvironment confers protection from chemotherapy induced apoptosis for a number of hematological malignancies, including AML. The majority of human AML blasts express VLA-4, the α4β1 integrin through which hematopoietic cells bind to VCAM-1 and/or fibronectin within the BM. Our laboratory and others have previously conducted in vitro studies demonstrating that antibody to VLA-4 enhanced chemotherapy induced cytotoxicity and apoptosis for AML cell lines and primary human AML blasts. One humanized anti-VLA-4 antibody (ab), natalizumab, is currently approved, with clinical applications in relapsing multiple sclerosis and Crohn’s disease, while a number of small molecule inhibitors of VLA-4 are under development with oral bioavailability for conditions such as asthma and inflammatory disorders. These oral agents would have the advantage of a shorter half-life than the humanized antibody, be available for just the period of chemotherapy treatment, and perhaps reduce the incidence of long-term toxicity. We herein present data that demonstrate
the ability of an oral small molecule inhibitor of VLA-4, D11-5908 (Daiichi Sankyo Co., Ltd.) to potentiate chemotherapy toxicity in AML blasts in vitro, comparable to anti-VLA-4 ab in this first direct comparison study, its ability to mobilize normal murine stem cells or engrafted AML cells in xenograft mice, and no impairment of blood cell recovery in vivo in normal mice receiving a combination of D11-5908 and ara-C compared to ara-C alone.
In independent experiments, the viability of AML blasts isolated from 8 patients pre-incubated with D11-5908, then treated with AraC (4 μM) or a combination of AraC (4 μM) and daunorubicin (5 μM), decreased by 27.8% ± 7.5% for cells on recombinant human fibronectin peptide CH-206, Retronectin™ (Rn), compared to a decrease of 10.4% ± 6.5% after pre-incubation with isotype control ab (p=0.0046 by two tailed paired t-test). This effect with D11-5908 was similar to the reduction in cell survival with the anti-VLA-4 ab, which decreased viability after chemotherapy by 20.2% ± 7.8% (p=0.014 compared to isotype control, and p=0.27 compared to D11-5908). Antibody to VLA-4 has been demonstrated to mobilize normal hematopoietic stem cells in vivo, in mice, non-human primates, and humans, a function that would be considered fundamental to an active VLA- 4 inhibitor. To test the ability of D11-5908 to mobilize both normal and AML cells in vivo from the marrow into the blood, we assayed for mobilization in both normal mice and in a xenograft model of human AML engrafted in NOD-scid β2microglobulin−/− mice. D11- 5908 mobilized both human AML cells, as well as normal murine progenitor cells in the NOD-scid mouse model. Mobilizing the human AML cells may render them susceptible to chemotherapy outside the protected BM microenvironment. Two of 4 NODscid mice previously engrafted with human AML cells mobilized human CD117 positive AML cells up to 50–60% of the peripheral blood mononuclear cells after treatment with D11- 5908, compared to negligible circulating AML prior to D11-5908 treatment. In addition, 2 untreated NODscid β2microglobulin−/− mice increased circulating murine colony forming cells (CFCs) from 253 ± 31/ml to 542 ± 106/ml of peripheral blood after three doses of oral gavage with D11-5908 (p=0.059). Four normal BALB/c mice, increased CFCs from 233/ml ± 14 to 471/ml ± 273 at 1 hour post 3rd dose of twice daily oral gavage with D11-5908. Lastly, D11-5908 did not impair normal blood cell recovery after AraC for BALB/c mice treated simultaneously with D11-5908 (100 mg/kg twice daily X 3 days) and AraC (20 mg/mouse IP), as compared to AraC alone in 5 independent experiments. The values for neutrophil count (ANC), nadir d.5-0.63 vs. 0.88 (p=0.30), recovery d.7-1.9 vs. 2.4 (p=0.13), and for a second experiment, nadir d. 3 -0.89 vs. 1.37 (p=0.095), recovery d. 5-1.23 vs.1.00 (p=0.38) did not differ significantly. The p values greater than 0.05 indicate that the blood cell recovery from nadir was equivalent for ara-C with or without D11-5908, as it was for white blood cells and hemoglobin; platelet counts were unaffected by these doses. These preclinical in vitro and in vivo data support the development of a promising therapeutic approach consisting of the combination of a novel oral adhesion inhibitor with chemotherapy for the treatment of AML.
Deubiquitination of Tip60 by USP7 Determines the Activity of the p53-Dependent Apoptotic Pathway
