NF449 Is a Novel Inhibitor of Fibroblast Growth Factor Receptor 3 (FGFR3) Signaling Active in Chondrocytes and Multiple Myeloma Cells

Abstract

The FGFR3 receptor tyrosine kinase represents an attractive target for therapy due to its role in several human disorders, including skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas. By using molecular library screening, we identified a compound named NF449 with inhibitory activity toward FGFR3 signaling. In cultured chondrocytes and murine limb organ culture, NF449 rescued FGFR3-mediated extracellular matrix loss and growth inhibition, which represent two major cellular phenotypes of aberrant FGFR3 signaling in cartilage. Similarly, NF449 antagonized FGFR3 action in the multiple myeloma cell lines OPM2 and KMS11, as evidenced by NF449-mediated reversal of ERK MAPK activation and transcript accumulation of CCL3 and CCL4 chemokines, both of which are induced by FGFR3 activation. In cell-free kinase assays, NF449 inhibited the kinase activity of both wild type and a disease-associated FGFR3 mutant (K650E) in a fashion that appeared non-competitive with ATP. Our data identify NF449 as a novel antagonist of FGFR3 signaling, useful for FGFR3 inhibition alone or in combination with inhibitors that target the ATP binding site.FGFR3 (fibroblast growth factor receptor 3) is a transmembrane tyrosine kinase that serves as a receptor for the members of the fibroblast growth factor (FGF) 2 family and functions in many biological processes, including cell proliferation, differentiation, migration, and survival. To date, activating mutations in FGFR3 have been associated with several human disorders, such as skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas (1-4).Among the skeletal dysplasias, activating FGFR3 mutations cause achondroplasia, the most common form of human skeletal dysplasia, and thanatophoric dysplasia, the most common form of lethal skeletal dysplasia (1). Long bones of individuals suffering from FGFR3-related skeletal dysplasias show markedly shortened zones of chondrocyte proliferation and differentiation, whereas Fgfr3 knock-out mice and humans without functional FGFR3 demonstrate skeletal overgrowth, together implying the role of FGFR3 as a negative regulator of bone growth (5).Apart from cartilage, ϳ15% of patients suffering from multiple myeloma markedly up-regulate FGFR3 as a consequence of a t(4;14)(p16.3;q32) translocation, with a fraction of patients also harboring activating mutations in FGFR3, identical to those found in the skeletal dysplasias (2, 3). Ectopic expression of FGFR3 enhances multiple myeloma cell proliferation and survival, demonstrating the oncogenic potential of FGFR3 (6).Given its role in human disease, FGFR3 signaling represents an attractive target for therapy. There is no treatment available for achondroplasia at present, thus inciting the development of novel approaches to target FGFR3. The aim of this study was to identify novel inhibitors of FGFR3 signaling in cellular environments relevant to FGFR3-related disorders. We recently established a chondrocyte cell-based reporter assay suitable for identification of inhibitors of ...

Novel therapies in MM: from the aspect of preclinical studies

“…More recently, a selective P2X 1 receptor antagonist NF449 has been shown to block FGFR3 signaling in MM cells. Importantly, NF449 inhibits the kinase activity of disease-associated FGFR3 mutant (K650E) in a fashion that is non-competitive with ATP [17]. Most recent studies show that a novel SM JAK inhibitor, AZD1480, blocks proliferation and survival, associated with inhibition of FGFR3 and JAK/STAT3 signaling in MM cells, cultured alone or with BMSCs [18].…”

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

“…This mutation has been exploited in many studies to examine signaling pathways utilized by FGFR3 (14,(39)(40)(41)(42)(43). However, the K650E mutation has not yet been identified together with the FGFR3-TACC3 oncogenic gene fusion in human cancer, and thus represents an artificial construct not yet found in nature.…”

Peripherally derived FGF21 promotes remyelination in the central nervous system

“…The BrdU incorporation activity was almost completely abolished by heat or Pronase treatment ( Figure 2B), indicating that the OPC proliferation factor in adult mouse serum must be a heat-sensitive protein. A pharmacological screen revealed that 2 FGF receptor (FGFR) inhibitors, PD173074 and NF449 (30,31), blocked serum-promoted OPC proliferation ( Figure 2, C and D, Supplemental Figure 2C, and Supplemental Table 1), suggesting that the OPC proliferation factor in adult mouse serum belongs to the FGF family. We then sought to determine which FGFRs are involved in serum-promoted OPC proliferation.…”