Targeting Lymphoplasmacytic Lymphoma through a Novel Anti-FGF-Based Therapeutical Strategy

INTRODUCTION. Bone marrow (BM) angiogenesis plays a crucial role in several hematologic malignancies, including both myeloid neoplasms and lymphoproliferative disorders. Nevertheless, the contribution of angiogenesis in patients with lymphoplasmacytic lymphoma/Waldenstrom's Macroglobulinemia (WM) and how it might be targeted for potential therapeutical intervention has not been described.

METHODS. Publically available mRNA profiling datasets of primary bone marrow (BM)-derived CD19 WM cells were used (i.e. GSE6691; GSE9656). The Gene Set Enrichment Analysis (GSEA) computational method was adopted (FDR<0.25; P<0.05). Co-culture proliferation and adhesion assays were performed using available WM (BCWM.1; MWCL1; WMWSU), IgM secreting cell lines (MEC1), and primary WM bone marrow (BM)-derived mesenchymal stromal cells (MSCs). Cell viability of primary CD19 positive-selected BM WM cells and peripheral blood-derived mononuclear cells (PBMC) was assessed by MTS assay. Modulation of signaling pathways was studied at protein level using western blot analysis. A pan-FGF trap NSC12 molecule was identified by a virtual screening of a NCI small molecule library: it acts a trap able to block the FGF2/FGFR interaction. NSC12 was tested in order to define its potential anti-WM activity in vitro.

RESULTS. GSE6691 gene expression dataset was used to investigate the most differentially expressed mRNA in WM cells as compared to their normal cellular counterpart: the top two pathways (ranked based on the normalized enrichment score and p-value) were related to "TNF-α signaling via NF-kB" and "angiogenesis". It has been already reported on the constitutive activation of NF-kB in WM; and therefore selected the "angiogenesis cascade" for further investigation (FDR 0.10; P 0.03). FGFR-1 was one of the most up-regulated genes in WM cells. We therefore looked at FGF- and FGFR-related mRNAs, using an independent GEP data set (GSE9656); and found FGFR-1, FGF-2, -12, -17 and -18 to be significantly enriched in WM cells as compared to their normal cellular counterpart (P<0.05). We next sought to determine the functional sequelae induced by the NSC12-mediated FGF2/FGFR blockade on WM cells. NSC12 exerted a direct anti-WM activity against all the cell lines tested (IC50: 3mM; P <0.05). Importantly, NCS12-induced cytotoxicity was demonstrated using WM patients' BM-derived CD19+ cells. NSC12 selectively targeted the FGF/FGFR axis, as demonstrated by inhibition of phospho(p)-FGFR1, as well as of downstream targets such as p-FSR2, p-JAK2, p-STAT3, and p-ERK. Of note, NSC12 inhibited WM cell proliferation even in a co-culture system with primary WM BM-MSCs. BM-MSC-induced up-regulation of p-FGFR1, p-STAT3, p-AKT was inhibited in WM cells exposed to increasing NCS12 doses, thus suggesting the activity of the compound even in the presence of the supportive BM milieu. We further confirmed NSC12-dependent induction of pro-apoptotic pathways, as shown at protein level by enhanced caspase-3- and PARP-cleavage. Notably, NSC12 did not induce cytotoxicity against healthy donor-PBMC.

CONCLUSIONS. These findings provide novel insights into the pathogenesis of WM, describing the up-regulation of the FGF/FGFR axis in WM cells, thus providing the preclinical rational for targeting FGF in this disease. Consistently, the novel pan-FGF trap NSC12 demonstrated an anti-WM activity, through both a direct and an indirect effect against WM cells and the WM cell/BM interaction, respectively.