Identification of activating mutations of the fibroblast growth factor receptor 4 in rhabdomyosarcoma

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Fibroblast growth factor receptor 4 (FGFR4), a tyrosine receptor kinase, is consistently over expressed in the pediatric malignancy rhabdomyosarcoma (RMS) suggesting a role in the tumor biology of RMS . FGFR4, along with other FGFR family members, regulate cell survival, motility and angiogenesis, and are frequently mutated in cancer. Our prior work has shown that FGFR4 mRNA levels correlate well with protein expression in RMS. Prior genetic studies have shown that a common FGFR4 transmembrane domain missense polymorphism (G388R) is associated with increased tumor cell motility and prognosis in patients with sarcomas and breast cancer. Based upon these studies we hypothesized that altered FGFR4 expression and function is due to the presence of mutations influencing RMS development and prognosis. To test this hypothesis, all FGFR4 coding exons were sequenced from 56 RMS tumor DNA samples. Twenty-seven single nucleotide variants were identified, of which 37% were present in a single tumor. In addition, 60% (16 of 27) were located in exons and 33% were nonsynonymous base substitutions. Six of the missense mutations (17.3% of 56 tumors) occur within functional domains with 4 (9.7%) occurring within the protein tyrosine kinase (PTK) domain. These PTK domain variants represent true mutations as none were present among 1030 healthy controls, although one control was a heterozygote for R529Q. Overall, PTK domain missense alleles were present at a higher prevalence in tumor DNA (n=5, 8.9%) than in healthy controls (n=1, 0.1%; P=1.8 x 10^-6), although it is not known if these are somatic or germline mutations. Predictive analysis of the PTK domain mutations suggests they disrupt protein function, which is consistent with their presence adjacent to the ATP binding pocket within FGFR crystal structures. These mutations are particularly intriguing as the paralogous codons of FGFR1, FGFR2, FGFR3, and RET are also mutated as either germline or somatic mutations in a diverse spectrum of diseases, including gliobastoma, Crouzon syndrome, hypochondroplasia, and familial medullary thyroid carcinoma. To examine the function of these PTK domain mutations in vitro, we used murine RMS cell lines that have previously been reported to have low metastatic potential and have undetectable FGFR4 protein levels. These lines were transfected with either wild type human FGFR4 or selected FGFR4 mutants. Transfectants expressing either FGFR4 N535K or V550E had a significant increase in cell proliferation and were less sensitive to the effects of serum free medium. These findings suggest that these FGFR4 PTK domain mutations are important for activating RMS cell growth. The identification of functional FGFR4 mutations adds to accumulating evidence that the FGFR signaling pathway is frequently mutated in human cancers and that agents targeting FGFRs might be broadly applicable as therapeutic candidates in oncology.