In Vivo Targeting of Stromal-Derived Factor-1 As a Strategy to Prevent Myeloma Cell Dissemination to Distant Bone Marrow Niches

Multiple myeloma (MM) patients present with multiple lytic lesions at diagnosis, indicating the presence of continuous dissemination of MM cells from the primary site of tumor development to multiple distant bone marrow (BM) niches. We hypothesized that stromal-derived factor-1 (SDF-1) may represent a target for preventing transition from MGUS (micrometastatic stage) to active-MM (macrometastatic stage); thus resulting in inhibition of MM progression. We therefore evaluated SDF-1 expression in the BM of patients with MGUS, MM, compared to healthy individuals; and tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1 in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vitro and in vivo .

SDF-1 levels were evaluated by immunohistochemistry on BM specimens obtained from patients with MGUS, active-MM, or healthy individuals; and confirmed by ELISA, using conditioned-medium of BM-mesenchymal stromal cells obtained from MGUS, active-MM and healthy individuals. BM metastatic lesions from primary epithelial tumors were also considered. Co-localization of MM tumor cells (MM.1S-GFP+) with SDF-1 was tested in vivo by in vivo confocal microscopy, using both AlexaFluor633-conjugated-anti-SDF-1 monoclonal antibody and AlexaFluor647-conjugated-NOX-A12 oligonucleotide. Effect of NOX-A12 on modulating MM cell dissemination was tested in vivo, by using in vivo confocal microscopy. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12+bortezomib. Detection of mobilized MM-GFP+ cells ex vivo was performed by flow cytometry. Effects of drug combination on dissemination of MM cells to distant BM niches was evaluated ex vivo by immunofluorescence on femurs obtained from each cohort of mice. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–100nM) treated primary MM BM stromal cells (BMSCs) in presence or absence of bortezomib (2.5–5nM) were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software. NOX-A12-dependent-modulation of signaling was evaluated by western blot on MM cells exposed or not to primary BM-MSCs.

Patients with active-MM present with higher BM SDF-1 expression vs. MGUS patients and healthy individuals. Similarly, BM presenting with metastasis from epithelial primary malignancies had higher SDF-1 levels compared to healthy subjects, thus suggesting the importance of SDF-1 in favoring tumor cell metastasis to BM niches. SDF-1 co-localized at BM level with MM tumor cells in vivo. In vitro, NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BMSCs supported by inhibition of BM-MSC-mediated phosphorylation of ERK1/2 and cofilin. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the peripheral blood of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). This was supported by inhibited homing of MM cells to the BM of those mice pre-treated with NOX-A12. We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 and bortezomib was observed (C.I.: .57-.76). These findings were validated in vivo: tumor burden was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly, significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 and bortezomib was observed, compared to bortezomib alone-treated mice (P <.05). Similarly, NOX-A12+bortezomib combination induced significant inhibition of MM cell homing, as shown by in vivo confocal microscopy.

SDF-1 represents a valid target for inhibiting MM cell dissemination to distant BM niches, thus providing the evidence for using the SDF-1 inhibiting Spiegelmer NOX-A12 to target MM cells at the stage of micrometastasis (MGUS), thus preventing development of symptomatic macrometastatic MM.

Zboralski:NOXXON Pharma AG, Berlin, Germany: Employment. Kruschinski:NOXXON Pharma AG, Berlin, Germany: Employment. Ghobrial:Novartis: Advisory Board Other; Onyx: Advisory Board, Advisory Board Other; Millennium: Advisory Board, Advisory Board Other; Bristol Myers Squibb: Advisory Board, Advisory Board Other.