Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): comparative analysis of Y373C, K650E and the novel G384D mutations

The t(4;14)(p16.3;q32) chromosomal translocation occurs in approximately 20 %, of multiple myelomas (MM) and leads to the apparent deregulation of two genes located on 4p16.3: the fibroblast growth factor receptor 3 (FGFR3) an d the putative transcription factor WHSC1/ MMSET, Interestingly, FGFR3 muta tions known to be associated with autosomal dominant human skeletal disorde rs have also been found in some MM cell lines with t(4;14) but their pathog enetic role in MM is still controversial, Since cell lines may represent us eful models for investigating the effects of deregulated FGFR3 mutants in M M, we analysed the expression, activation, signaling pathways and oncogenic potential of three mutants identified so far: the Y373C and K650E in the K MS-11 and OPM-2 cell lines respectively, and the novel G384D mutation here identified in the KMS-18 cell line, All of the cell lines present a heteroz ygous FGFR3 gene mutation and transcribe the mutated allele; unlike KMS-11 and OPM-2 (which express the IIIc isoform), the KMS-18 cell line expresses prevalently the isoform IIIb, We demonstrated that, under serum-starved con ditions, KMS-11 and OPM-2 cells express appreciable levels of phosphorylate d FGFR3 mutants indicating a constitutive activation of the Y373C and K650E receptors; the addition of the aFGF ligand further increased the level of receptor phosphorylation, Conversely, the FGFR3 mutant in KMS-18 does not s eem to be constitutively activated since it was phosphorylated only in the presence of the ligand, In all three MM cell lines, ligand-stimulated FGFR3 mutants activated the MAP kinase signaling pathway but did not apparently involve either the STAT1 or STAT3 cascades, However, when transfected in 29 3T cells, G384D, like Y373C and K650E, was capable of activating MAPK, STAT 1 and STAT3 under serum-starved condition, Finally, a focus formation assay of NIH3T3 cells transfected with FGFR3-expressing plasmid vectors showed t hat Y373C and K650E (albeit at different levels) but not G384D or the wild- type receptor, can induce transformed foci, Overall, our results support th e idea that FGFR3 mutations are graded in terms of their activation capabil ity, thus suggesting that they may play a critical role in the tumor progre ssion of MM patients with t(4;14).