Bortezomib Attenuates Adhesion of B Cell Precursor Acute Lymphoblastic Lleukemia Cells to Bone Marrow Mesenchymal Stromal/Stem Cells Via Regulating SPARC Expression

The prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is poor. Many cases in complete remission experience relapse of leukemia despite intensive chemotherapy or hematopoietic stem cell transplantation. This clinical observation suggests that minimal residual disease (MRD) still exists after these intensive therapies. Cell adhesion-mediated drug resistance (CAM-DR) is one of the mechanisms to support MRD in the bone marrow microenvironment (Clin Cancer Res 14:9, 2008). Mesenchymal stromal/stem cells (MSCs) are a cellular component of bone marrow (BM) and maintain physiological precursor B lymphopoiesis (Nature Rev Immunol 6:107, 2006). We investigated our hypothesis that survival of BCP-ALL cells is supported by their direct adhesion to BM-MSCs in BM microenvironment.

First, we confirmed that BCP-ALL cells exhibited their drug resistant phenotype through adhesion to human BM-MSCs using human BCP-ALL cell line, Nalm6. We isolated human BM-MSCs from normal bone marrow samples (AllCells, Emeryville, CA). When Nalm6 cells were co-cultured with human BM-MSCs, they were prone to adhere to BM-MSCs. Nalm6 adhered to BM-MSCs (Nalm6/ad) were more resistant to the treatment with various anti-cancer drugs including doxorubicin than Nalm6 in suspension (Nalm6/su). Immunoblot analysis showed that expression level of anti-apoptotic protein Bcl-2 and phosphorylation level of pro-survival kinase Akt are higher in Nalm6/ad compared with those in Nalm6/su. In cell cycle analysis using Ki67/PI fluorescence-activated cell sorter (FACS) assay, the percentage of populations in G0 phase and S/G2/M phase was higher in Nalm6/ad compared with that in Nalm6/su, which indicated the increase of MRD and the high proliferation of leukemic cells in adhesive population, respectively. Therefore, we considered that detachment of Nalm6 from BM-MSCs is important to restore chemosensitivity of BCP-ALL cells and to eliminate these cells.

Accordingly, we screened drugs that are capable of disrupting the adhesion of Nalm6 to BM-MSCs, and found that several proteasome inhibitors had a such activity. BM-MSCs were treated with bortezomib (10nM for 24h or 100nM for 1h), carfilzomib (3 nM for 24 h or 100 nM for 1 h) or oprozomib (10 nM for 24 h or 300 nM for 4 h), and then washed with PBS and co-cultured with Nalm6 cells. We confirmed that the number of BM-MSCs is not affected with these drugs irrespective of their concentration and incubation time by MTT assay. Co-cultured cells were divided into suspension cells and adherent cells. Each population was stained with monoclonal antibodies against CD19 and CD90 to detect Nalm6 and BM-MSCs, respectively, and analyzed by FACS. When BM-MSCs were pre-treated with bortezomib, carfilzomib or oprozomib, the number of Nalm6/ad was significantly decreased, and inversely, the number of Nalm6/su was increased, as compared to that of co-cultures with untreated BM-MSCs.

Intriguingly, BM-MSCs treated with bortezomib showed aberrant expression of secreted protein acidic and rich in cysteine (SPARC). Immunoblot analysis showed that SPARC expression of BM-MSCs was transiently increased after bortezomib treatment. In co-cultures with BM-MSCs transfected with siRNA targeting SPARC, the number of Nalm6/ad was increased. In addition, the number of Nalm6 adhered to BM-MSCs pre-treated with recombinant human SPARC was lower than that adhered to control non-treated BM-MSCs. Therefore, SPARC showed anti-adhesive property and was one of the molecules associated with adhesion of BM-MSCs to Nalm6.

Finally, we tested whether bortezomib shows therapeutic effects in vivo. Bortezomib administration supported survival of BCP-ALL xenograft model mice utilizing Nalm6, which was concomitant with the decrease in leukemia cells in the femur and in the size of spleen, as compared to those in non-treated control mice.

In summary, Nalm6 cells that adhered to BM-MSCs contributed to construction of chemoresistant population similar to MRD. Here, we found that this population could be detached by treatment of BM-MSCs with bortezomib through transient increase in their SPARC expression. Our findings suggest that bortezomib or other proteasome inhibitors might be promising drugs to treat BCP-ALL through attenuating the adhesion of leukemic cells to BM-MSCs, and shed new insight into a therapeutic strategy in BCP-ALL by targeting BM-MSCs.