Inhibition of αvβ3 Integrin Disrupts Adhesion and Enhances Chemotherapy Effect in Multiple Myeloma Cells.

Abstract

Multiple myeloma cells adherent within the bone marrow microenvironment exhibit protection from chemotherapy. Myeloma cells express the αvβ3 receptor for fibronectin (Fn) and vitronectin (Vn), a receptor critical for tumor invasiveness and tumor angiogenesis. Disruption of angiogenesis or interference with adhesion of myeloma cells inhibits myeloma growth. Human myeloma cell line U266 exhibited 41% expression of αvβ3 integrin by flow cytometry using LM609 monoclonal antibody (Chemicon) or 82% using MEDI-522 humanized antibody (Abegrin™, kindly provided by Medimmune Oncology, Inc. and the National Cancer Institute). For bone marrow mononuclear cells from a patient with multiple myeloma, 99.5% of the CD38 positive myeloma cell fraction labeled with MEDI-522 antibody. Multiple myeloma cells exhibited resistance to doxorubicin when grown on fibronectin, vitronectin, or HS27a stromal cells (the latter generously provided by Dr. B. Torok-Storb). Antibody to αvβ3 resulted in enhanced cell kill and apoptosis by doxorubicin when cells were grown on matrix proteins or stromal cells. U266 cells were incubated with αvβ3 blocking antibody, or isotype control for one hour, before being plated on Fn, Vn, or on bovine serum albumin (BSA) as a control. Cells were then treated with 20μM doxorubicin, or no drug, for 16 hours at 37°C. Viability was determined by trypan blue exclusion, and apoptosis was assayed by Annexin-V and caspase-3 activity. Multicolor flow cytometry was performed with FITC labeled CD38 antibody to identify the myeloma cells, as well as antibodies to detect annexin V and caspase 3. On Fn, Vn, or stromal cell line HS27a, αvβ3 function blocking antibody (LM 609) reduced cell viability and increased apoptosis (increased annexin V and caspase 3 staining). TABLE 1: LM609 Antibody in U266 Cells On Fibronectin Viability Annexin V Caspase 3 Annexin V and Caspase 3 staining are measures of apoptosis Isotype control 74% 4% 15% αVβ3 antibody 22% 49% 60% On Vitronectin Isotype control 65% 12% 45% αVβ3 antibody 20% 54% 74% On stromal cell line Isotype control 65% 39% 28% αVβ3 antibody 15% 97% 97% In addition, HS-27a stromal cells were plated in wells, and U266 cells were added with or without pre-incubation with αvβ3 blocking antibody, or isotype control, then doxorubicin was added. Myeloma cells plated on the stromal cells exhibited chemoresistance, compared to the BSA control, while antibody to αvβ3 restored cytotoxicity and apoptosis. MEDI-522 antibody reduced binding of U266 cells to fibronectin by 78%, and similar to results with the LM609 antibody, enhanced chemotherapy response on Fn or Vn, with reduction of cell viability and increase in apoptosis. Table 2: MEDI-522 Antibody in U266 Cells On Fibronectin Viability Annexin V Isotype control 64% 51% αVβ3 antibody 48% 59% On Vitronectin Isotype control 68% 38% αVβ3 antibody 48% 54% Each flow cytometry result represents 10,000 events; therefore, the differences between the anti-αvβ3 antibody vs. isotype control are statistically significant. Inhibition of αvβ3 integrin is thus able to overcome adhesion mediated chemotherapy resistance in myeloma cells. The Abegrin™ antibody to αvβ3 has undergone early phase trials in cancer patients, and we and others have shown that myeloma patient cells exhibit a high fraction of staining with this antibody. These preclinical studies support the novel application of Abegrin™ for the treatment of multiple myeloma.