Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors

Abstract

The Ras/MAPK pathway is critical for human development and plays a central role in the formation and progression of most cancers. Children born with germ-line mutations in BRAF, MEK1 or MEK2 develop cardio-facio-cutaneous (CFC) syndrome, an autosomal dominant syndrome characterized by a distinctive facial appearance, heart defects, skin and hair abnormalities and mental retardation. CFC syndrome mutations in BRAF promote both kinase-activating and kinase-impaired variants. CFC syndrome has a progressive phenotype, and the availability of clinically active inhibitors of the MAPK pathway prompts the important question as to whether such inhibitors might be therapeutically effective in the treatment of CFC syndrome. To study the developmental effects of CFC mutant alleles in vivo, we have expressed a panel of 28 BRAF and MEK alleles in zebrafish embryos to assess the function of human disease alleles and available chemical inhibitors of this pathway. We find that both kinase-activating and kinase-impaired CFC mutant alleles promote the equivalent developmental outcome when expressed during early development and that treatment of CFC-zebrafish embryos with inhibitors of the FGF-MAPK pathway can restore normal early development. Importantly, we find a developmental window in which treatment with a MEK inhibitor can restore the normal early development of the embryo, without the additional, unwanted developmental effects of the drug.

Figure 1.

CFC syndrome alleles promote developmental changes during early embryogeneis. (A) RNA expression of CFC and melanoma variants BRAF^Q257R (kinase-activating, CFC), BRAF^G596V (kinase-impaired, CFC and melanoma) and BRAF^V600E (very high-kinase, melanoma) cause elongation of the developing zebrafish embryo at 12 hpf, and severe developmental abnormalities at 24 and 48 hpf. In contrast, embryos expressing BRAF^WT undergo normal development at all stages. No differences were detected in WT or disease allele expressing 4 hpf embryos. (B) Western blotting of zebrafish extracts reveals expression of the myc-tagged BRAF variants with the 9E10 antibody, and α-tubulin is a loading control.

Figure 2.

Analysis and treatment of kinase-activating and kinase-impaired BRAF and MEK disease variants. (A) RNA expression of BRAF CFC and melanoma variants, as well as engineered BRAF mutations, promotes an elongated embryo phenotype. RNA expression of wild-type BRAF has no effect on development. The developmental phenotypes caused by the BRAF variants are prevented by treatment with the MEK inhibitor, CI-1040 and the embryo has a normal rounded shape. (B) Western blotting of zebrafish lysates with anti-ERK and anti-phospho-ERK antibodies reveals a reduction in the ratio of phospho-ERK to total ERK protein in treated embryos, and α-tubulin is a loading control. (C) RNA expression of CFC MEK variants, the MEK1 constitutively activating (ΔN3DD) and the MEK2 kinase-inactivating K101M mutations promotes elongation in the developing embryo. Expression of wild-type MEK1 and MEK2 do not affect development. The developmental phenotypes caused by the MEK alleles are prevented by treatment with the MEK inhibitors, CI-1040 and PD0325901. (D and E) Western blotting of zebrafish lysates with anti-ERK and anti-phospho-ERK antibodies reveals downstream activity of phospho-ERK and confirms the potency of the MEK inhibitors, and α-tubulin is a loading control.

Figure 3.

BRAF and MEK disease alleles promote cell movement phenotypes. (A) The most common CFC varient, BRAF^Q257R, was expressed in developing embryos, and the embryos imaged every hour. Development of the embryos begins to become affected by BRAF^Q257R expression between 7.5 and 8.5 hpf. (B) BRAF and (C) MEK disease variants promote significant changes in cell movement, as revealed by in situ hybridization. The gene expression domain of dlx3 is altered for both expression pattern and intensity of signal, such that there is no dlx3 detected in the BRAF^V600E expressing embryos. HggI expression remains unaffected. The bottom panel shows the lateral view of the same embryos.

Figure 4.

BRAF kinase-active and kinase-impaired alleles can promote an additive effect during development. Embryos co-expressing combinations of suboptimal doses (15 pg) of kinase-active (BRAF^Q257R, BRAF^S467A) and kinase-impaired (BRAF^G596V) CFC alleles or BRAF^WT mRNA were assessed for the elongation phenotype at 10 hpf. The number of elongated embryos did not change significantly upon expression of a single BRAF CFC allele (15 pg), or in combination with BRAF^WT (for a total of 30 pg). A significant increase in the mutant phenotype was induced by co-injections of BRAF^Q257R with BRAF^S467A (P < 0.0001) or BRAF^Q257R with BRAF^G596V (P = 0.0003) compared with the BRAF CFC allele co-injected with BRAF^WT as indicated by χ² tests. The numbers in the bars indicates the percentages of elongated embryos; n is the number of injected embryos.

Figure 5.

Identification of a zebrafish-CFC treatment window. (A) Six CI-1040 treatments (A–F) and a control treatment (G) for zebrafish embryos expressing CFC variant BRAF^Q257R reveal that all drug treatments restore normal development at 10.5 hpf (treatments A–F), whereas only treatments A and B restore normal development both at 10.5 and 27.5 hpf. Other drug treatments (C–F) cause additional developmental defects at 27.5 hpf, most notably, reduced posterior development. (B) Six additional drug treatments (A–F) and a control treatment (G) for zebrafish-CFC variant BRAF^Q257R show that the 4.5–5.5 hpf developmental window is necessary for the prevention of zebrafish-CFC early phenotypes. The percentage of embryos displaying the phenotype is given at the lower right of each image (a minimal of n = 30/experiment). Blue bars represent embryo medium, whereas red bars represent CI-1040 treatment.

Figure 6.

Treatment of BRAF, MEK1 and MEK2 variants with SU-5402. (A) The mutant phenotypes promoted by the RNA expression of BRAF variants are prevented by pharmacological treatment with the FGFR1 inhibitor SU-5402, with the exception of the developmental phenotype caused by BRAF^V600E. (B) Similarly, the elongation promoted by MEK1 and MEK2 disease variants is prevented by SU-5402 treatment. (C) Western blotting of total zebrafish lysates for ERK and phospho-ERK protein shows that SU-5402 treatment causes reduction of ERK phosphorylation, with α-tubulin as a loading control.

Figure 7.

Evaluation of CFC-disease variant in vivo activity and potential treatment. Schematic representation of the zebrafish-based approach designed to examine the in vivo significance of BRAF and MEK CFC disease mutations. (A) Microinjection of BRAF or MEK CFC variant mRNA into the single-cell zebrafish embryo promoted an elongated zebrafish embryo at 10 hpf that gives rise to an animal with severe development defects, including axis formation, and heart defects at 24 hpf (red arrow). (B) Treatment of CFC-microinjected embryos with inhibitors of the FGF-MAPK signalling pathway (green) restores normal development to the CFC-zebrafish embryo, possibly by restoring appropriate total levels of MAPK-signalling (green arrows).