Our study addresses the in vivo
action of CFC mutant alleles and may point to a potential therapeutic approach for individuals with CFC syndrome. First, we have demonstrated that CFC mutant alleles cause similar developmental phenotypes in an in vivo
zebrafish model system, despite their in vitro
kinase activity. Second, we have used our model system to explore the therapeutic potential of small molecule inhibitors to prevent the in vivo
activity of CFC mutations during early development. We have evaluated both the developmental activity and the therapeutic potential of 18 human CFC and three melanoma disease alleles, as well as three different small molecule inhibitors, in 12 treatment conditions. In this work, zebrafish embryos are injected at the single cell stage with RNA of the human disease allele, or with control RNA, and the phenotype of the embryo assessed by 10 h (Figs , and A). Embryos normally express FGF-MAPK signalling during development in a localized manner to shape the development of the embryos during gastrulation (18
). We found BRAF and MEK kinase-active and kinase-impaired disease variants interfere with convergence–extension cell movements during gastrulation (Fig. ), providing insight into how similar clinical CFC phenotypes are caused by kinase-activating and kinase-impaired alleles. Future studies will reveal how the effects on early cell movement (Fig. ) correlate with disease allele penetrance and disease presentation in humans.
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 (more ...)
In our in vivo
animal system, and in the context of endogenous signalling, we find CFC alleles with kinase-inactivating mutations, as defined in vitro
, promote the same phenotype as kinase-active alleles. One possibility is that BRAF kinase-impaired proteins interact with CRAF to stimulate MEK-ERK signalling (9
). Kinases frequently act through dimerization, including BRAF and CRAF (10
), and crystal structures of MEK predict MEK1 and MEK2 self associate via a homodimerization interface to form stable dimers (37
). Such mechanisms may be at work in our zebrafish studies, providing the molecular context for kinase-impaired BRAF and MEK alleles to be able to promote active signalling of the pathway, including the engineered kinase-inactive alleles (36
) as determined by in vitro
kinase assays (Fig. ). Another possibility is that dysregulation of Ras/MAPK signalling through gain-of-function or loss-of-function mutations may cause similar disease phenotypes (38
). As an important example, the disease spectrum associated with varying SHP-2 mutations in Noonan syndrome and cancer argue against SHP-2 activity as the defining predictor of disease outcome (39
). Both loss-of-function and gain-of-function mutations in SHP-2 lead to the clinically similar LEOPARD and Noonan syndromes, and expression of LEOPARD and Noonan syndrome alleles in zebrafish and Drosophila
produce equivalent developmental phenotypes (15
). Our work suggests that both kinase-active and kinase-impaired CFC alleles are effectively gain-of-function mutations and activate the pathway because combinations of active and impaired BRAF mutant alleles can promote an additive effect during early development (Fig. ).
Designing new therapies for rare birth disorders is problematic due to the great costs and research efforts of drug development, and the required clinical safety and efficacy testing for new therapeutics (40
). Since the Ras/MAPK pathway has been a prime target for cancer therapeutics, application of these small molecule inhibitors presents a possible therapeutic avenue, since the underlying molecular dysfunction is common. Previously, the activity of CFC MEK alleles has been shown to be sensitive to MEK inhibitors in cells (12
). Direct testing of the effects of anti-cancer therapeutics on BRAF and MEK CFC characteristics in zebrafish is an important next step in exploring the therapeutic potential for CFC syndrome. Using our model, we have tested the ability of FGF-MAPK inhibitors to prevent the developmental effects of CFC and melanoma disease alleles (Fig. B). We found that MEK inhibitors prevent the cell migration defects caused by the disease alleles, and also that additional developmental side-effects of the drug could be avoided by treating the embryos within a specific developmental time-window (Fig. A). These results suggest that future studies in pre-clinical models of CFC should explore if similar drug treatment time windows may help ease the developmental abnormalities and symptoms associated with CFC progression. However, because CFC mutations affect gastrulation (Fig. –), and have an early developmental treatment window (Fig. ), application of MEK inhibitors for CFC syndrome patients may be severely limited. Nonetheless, because CFC syndrome has a progressive phenotype, and many of the phenotypic effects develop post-natally, patients may still be helped by systemic therapies after birth (1
We also provide evidence that the developmental effects of the disease alleles can be prevented by the inhibition of endogenous FGFR-signalling, with the exception of one of the highest kinase-activating melanoma mutations, BRAFV600E
(Fig. ). We reason that as normal gastrulation involves endogenous FGFR signalling, FGFR inhibition reduces the total level of defective CFC BRAF or MEK signalling, thereby preventing the altered cell movement phenotype. This supports the idea that total MAPK signalling is important in CFC development (Fig. ), and also emphasizes the importance of testing the action of developmental syndrome mutant alleles and inhibitors in a developing animal. In vitro
, the CFC BRAFQ257R
and melanoma BRAFV600E
mutant alleles both promote similar high-kinase activity (7
), and yet no individual with CFC syndrome has been identified with a BRAFV600E
mutation. This demonstrates, for the first time, that the BRAFV600E
mutation is probably stronger in vivo
than the CFC mutations.
The high conservation of the MAPK signalling pathway means that our CFC chemical-genetic studies in zebrafish embryos will be relevant to the development of future pre-clinical models of CFC. For example, mice exhibiting Apert-like syndrome from dominant mutations in fibroblast growth factor receptor-2 can be treated pre- and post-natally with the small molecule MEK inhibitor, U0126 (41
). We note, however, that similar comprehensive CFC allele comparisons, coupled with multiple treatment testing, within the short-time span described here, is not currently feasible in mouse models. This makes the zebrafish system a tractable tool for medical and research geneticists to explore allele activity and therapeutic potential. This work establishes a foundation to propel forward the clinical discussion and scientific strategy for assessing the suitability of using currently available cancer drugs to treat the progressive phenotypes of CFC in children.