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

Abstract

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 ®broblast growth factor receptor 3 (FGFR3) and the putative transcription factor WHSC1/ MMSET. Interestingly, FGFR3 mutations known to be associated with autosomal dominant human skeletal disorders have also been found in some MM cell lines with t(4;14) but their pathogenetic role in MM is still controversial. Since cell lines may represent useful models for investigating the e ects of deregulated FGFR3 mutants in MM, we analysed the expression, activation, signaling pathways and oncogenic potential of three mutants identi®ed so far: the Y373C and K650E in the KMS-11 and OPM-2 cell lines respectively, and the novel G384D mutation here identi®ed in the KMS-18 cell line. All of the cell lines present a heterozygous 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 conditions, KMS-11 and OPM-2 cells express appreciable levels of phosphorylated 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 seem 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 293T cells, G384D, like Y373C and K650E, was capable of activating MAPK, STAT1 and STAT3 under serum-starved condition. Finally, a focus formation assay of NIH3T3 cells transfected with FGFR3-expressing plasmid vectors showed that Y373C and K650E (albeit at di erent levels) but not G384D or the wild-type receptor, can induce transformed foci. Overall, our results support the idea that FGFR3 mutations are graded in terms of their activation capability, thus suggesting that they may play a critical role in the tumor progression of MM patients with t(4;14). Oncogene (2001) 20, 3553 ± 3562.

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

“…The nonfused tyrosine kinase domains (lanes 2, 3, and 4) were present mainly in the cytoplasmic fraction, which also includes the cell membrane fraction. Perinuclear localization of FGFR3 (K650E) has been demonstrated previously (37), but fusion of FGFR3(K650E) to TACC3 dramatically increased nuclear localization. These results indicate the presence of the TACC3 coiledcoil domain is responsible for nuclear localization of the FGFR3 kinase, regardless of receptor activation.…”

A Novel, Selective Inhibitor of Fibroblast Growth Factor Receptors That Shows a Potent Broad Spectrum of Antitumor Activity in Several Tumor Xenograft Models

“…5A) were collected and processed. This molecule is also effective against OPM-2, which overexpresses FGFR3 and is also known to contain an activating mutation in its kinase domain (44). In addition, this molecule is potent at inhibiting the in vivo growth of tumor xenografts derived from these diverse cancer cell lines.…”

Targeting FGFR3 in multiple myeloma: inhibition of t(4;14)-positive cells by SU5402 and PD173074

“…However, this line has been reported to express FGFR3 only very weakly, 19 and to harbour a Ras rather than a FGFR3 mutation. 9 This is consistent with recent data suggesting that up to 30% of IgH-MMSET-positive MM patients are FGFR3-expression negative 20,21 and explains the absence of an inhibitory effect by SU5402 or PD173074.…”