Tassone, Pierfrancesco

Abstract BAFF is a member of the tumor necrosis factor (TNF) family and plays a role in B cell survival including MM cells. We have previously established a role for BAFF in localization and survival of MM cells in the bone marrow (BM) microenvironment. (Cancer Res2006; 66(13): 6675–82). Here we validate the role of BAFF in MM patients and demonstrate the in vivo activity of anti-BAFF antibody in a SCID-hu MM model. We first performed gene expression profiling (GEP) on CD 138+ plasma cells isolated from 90 MM patients and 11 healthy controls using the Affymetrix U133A arrays. GEP analysis demonstrated increased BCMA expression (p<0.0001, student T test) on newly diagnosed and relapsed MM versus normal plasma cells. Flow cytometry performed on MM patient cells demonstrated the presence of all 3 receptors on CD 138+ cells. ELISA assays confirmed increased plasma BAFF levels in 51 MM plasma (mean: 1049 pg/ml; range: 176–4252 pg/ml) compared to 11 normal donors (mean: 461pg/ml; range: 317–652pg/ml) [p<0.001]. To understand the functional significance that BAFF might play in the biology of MM, we studied the effects of recombinant BAFF (rh-BAFF) on MM cells directly and in the context of its BM microenvironment. Our data demonstrate that rh-BAFF confers a survival advantage to MM cells and protects them against dexamethasone-induced cytotoxicity. Importantly, anti-apoptotic proteins Bcl2 and XIAP were upregulated, as were growth and survival signals belonging to the AKT and MAPKinase pathways. Because of the survival advantage conferred by BAFF on MM cells we evaluated the use of a clinical grade-neutralizing antibody to BAFF. To evaluate the in vivo activity of anti-Baff in MM we used the SCID-hu model, where MM cells grow in the context of human BM microenvironment. A cohort of SCID-hu mice bearing INA-6 MM cells, were treated i.p. weekly with anti-BAFF neutralizing antibody (10 mg/kg, n=3) or control isotype (10 mg/kg, n=3), respectively, for four weeks. Serum levels of soluble human IL-6 receptor (shuIL-6R) released by MM cells into the murine serum were monitored as a measure of MM growth. At the end of treatment we observed a significant (p=0.048) reduction in shuIL-6R level. This translated into a survival advantage of 3.1 weeks in the anti-BAFF treated animals versus the control group (p= 0.02). We also evaluated in vivo effects of anti-BAFF on the bone compartment by radiographic analysis and tartrate-resistant acid phosphatase (TRAP) staining of human bone implants. Our results demonstrate a decrease in radiologically evident lytic lesions in the anti-BAFF treated animals. This was accompanied by a significant decrease in TRAP + osteoclasts in bone sections from treated mice when compared to control mice. Our data therefore suggests that anti-BAFF antibody may impact MM cell growth directly and/or via effects on the BM microenvironment by impacting bone resorption. Taken together, these data show a role for BAFF mediating MM cell survival and its in vivo anti-tumor activities provide a preclinical rational for its clinical evaluation in MM.

Abstract NF-κB is a transcriptional factor promoting tumor cell growth and inhibition of apoptosis via regulating expression of proteins modulating cell cycle and anti-apoptosis. NF-κB is constitutively associated with an inhibitor (IκB), which is phosphorylated by IκB kinase (IKK) upon cell stimulation (ie, TNFα) and subsequently degraded by ubiquitin-proteasome pathway, thereby allowing NF-κB to translocate to nucleus. We have previously shown that an IKK inhibitor PS-1145 partially (20–50%) inhibits MM cell proliferation; however, it inhibits both IL-6 secretion from BMSCs triggered by MM cell adhesion and proliferation of MM cells adherent to BMSCs. Targeting IKKβ is therefore a possible therapeutic option for inhibition of MM cell growth in the bone marrow microenvironment by downregulating NF-κB activity. In this study, we further delineated the biologic significance of IKK inhibition in MM cells using IKKβ specific inhibitor MLN120B (Millennium Pharmaceuticals, Cambridge, MA). MLN120B induced 60–90% growth inhibition in cells from the MM cell lines RPMI8226, RPMI/Dox40, RPMI/LR5, U266, and INA-6; on the other hand, it induced only 25–30% inhibition in MM.1S and MM.1R cells, assessed by 72 h tritiated-thymidine uptake. Importantly, neither IL-6 nor IGF-1 overcomes the growth inhibitory effect of MLN120B in both MM.1S and U266 cells. Interestingly, MLN120B significantly augmented TNFα-induced cytotoxicity in MM.1S cells. We next examined whether MLN120B could enhance the cytotoxicity of other agents. MLN120B augmented growth inhibition triggered by doxorubicin and melphalan in RPMI8226 and IL-6 dependent INA-6 cell line. We therefore studied growth inhibitory effect of MLN120B in the presence of bone marrow stromal cells (BMSCs). MLN120B inhibited 70–80% of constitutive IL-6 secretion from BMSCs, without toxicity. Importantly, MLN120B almost completely blocked stimulation of MM.1S, U266 and INA-4 cell growth and IL-6 secretion from BMSCs induced by binding of tumor cells to BMSCs. Finally, MLN120B overcame the protective effect of BMSCs, cell adhesion mediated drug resistance, against dexamethasone in MM.1S cells. Taken together, our data demonstrate that an IKKβ inhibitor induces cytotoxicity in MM cells in the BM milieu, providing the framework for clinical trials of these novel agents to improve patient outcome in MM.