Abstract B134: Inhibition of the fibroblast growth factor system by a new FGF trap induces oxidative stress and mitochondrial apoptosis in multiple myeloma cells
Authors: Giacomini Arianna , Ghedini Gaia Cristina , Maccarinelli Federica , Locatelli Silvia Laura , Sacco Antonio , Castelli Riccardo , Desantis Vanessa , Roccaro Aldo Maria , Mor Marco , Carlo-Stella Carmelo , Ronca Roberto , Presta Marco 
Published: August 2018

Abstract
Despite advances in systemic and supportive therapies, multiple myeloma (MM) remains incurable because of chemotherapeutic resistance. Fibroblast growth factor-2 (FGF2) plays a pivotal role in MM by acting as an autocrine/paracrine mitogen on plasma cells, bone marrow-derived endothelial cells, and fibroblasts. In keeping with the pivotal role of the FGF system in MM, a recurrent chromosomal translocation t(4;14) is associated with upregulation of FGF receptor-3 (FGFR3) in MM patients. Agents able to hamper FGF signaling are therefore of interest as a novel approach for the treatment of MM. The soluble pattern recognition receptor long pentraxin-3 (PTX3) owns a unique FGF-binding N-terminal domain. Structural/functional characterization of this domain led to the characterization of the pentapeptide ARPCA as a potent FGF antagonist in vitro and in vivo. Subsequently, molecular modeling based on ARPCA structure and small-molecule library fishing have allowed the identification of a novel, orally active 480 Da FGF trap (NSC12) able to hamper tumor growth and vascularization in FGF-driven human lung and prostate tumor models. Here, the therapeutic potential of NSC12 for MM treatment was tested on four human MM cell lines harboring (KMS-11 and OPM-2 cells) or not (U-266 and RPMI8226 cells) the t(4:14) translocation. NSC12 blocked the proliferation of all human MM cell lines with an IC50 ≅ 3 µM. Western blot analysis showed that NSC12 inhibited the activation of FGFR3 signaling and strongly downregulated the antiapoptotic protein mcl-1, leading to the activation of caspase 3. Cytofluorimetric analysis revealed a significant increase of annexin-V+ apoptotic cells as early as 6 hrs after NSC12 treatment, with 100% of cell death after 24 hrs of treatment. Interestingly, apoptosis was observed even when MM cells were cocultured with patient-derived bone marrow stromal cells. In vivo, oral delivery of NSC12 significantly blocked the growth of KMS-11 (286.9 ± 22.4 vs 459.8 ± 27.4 mm3, p&lt;0.001) and RPMI8226 (141.5 ± 17.7 vs 245.9 ± 46.4 mm3, p&lt;0.001) xenografts injected subcutaneously in immune-compromised NOD/SCID mice. Analysis of tumor xenografts from NSC12-treated mice showed a strong increase of TUNEL-positive apoptotic cells and a significant reduction of CD31-positive tumor vascularization compared to xenografts from vehicle-treated mice. In an attempt to get further insights into the proapoptotic mechanism of action of NSC12 in MM cells, we found that NSC12 treatment rapidly induces a significant and progressive increase of mitochondrial depolarization paralleled by massive mitochondrial ROS production, suggesting a key role of mitochondria dysfunction in NSC12-induced MM cell death. In addition, as observed for MM cell lines, NSC12 treatment of patient-derived MM cells (N. of patients=14) strongly reduced FGFR3 phosphorylation and mcl-1 levels and induced the upregulation of oxidative stress-induced genes accompanied by apoptosis. Altogether, these data suggest that NSC12 affects MM tumor growth through a direct cytotoxic activity on MM cells and by inhibiting FGF-mediated paracrine/autocrine crosstalk in the tumor stroma/microenvironment. Thus, NSC12 may represent a novel “two compartment” inhibitor in MM by hampering FGF-dependent angiogenic/oncogenic signaling.
Citation Format: Arianna Giacomini, Gaia Cristina Ghedini, Federica Maccarinelli, Silvia Laura Locatelli, Antonio Sacco, Riccardo Castelli, Vanessa Desantis, Aldo Maria Roccaro, Marco Mor, Carmelo Carlo-Stella, Roberto Ronca, Marco Presta. Inhibition of the fibroblast growth factor system by a new FGF trap induces oxidative stress and mitochondrial apoptosis in multiple myeloma cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B134.