RAF1 deficiency causes a lethal syndrome that underscores RTK signaling during embryogenesis

Abstract

Somatic and germline gain-of-function point mutations in RAF, one of the first oncogenes to be discovered in humans, delineate a group of tumor-prone syndromes known as the RASopathies. In this study, we document the first human phenotype resulting from the germline loss-of-function of the proto-oncogene RAF1 (a.k.a. CRAF). In a consanguineous family, we uncovered a homozygous p.Thr543Met variant segregating with a neonatal lethal syndrome with cutaneous, craniofacial, cardiac, and limb anomalies. Structure-based prediction and functional tests using human knock-in cells showed that threonine 543 is essential to: (i) ensure RAF1's stability and phosphorylation, (ii) maintain its kinase activity toward substrates of the MAPK pathway, and (iii) protect from stress-induced apoptosis mediated by ASK1. In Xenopus embryos, mutant RAF1^T543M failed to phenocopy the effects of normal and overactive FGF/MAPK signaling, confirming its hypomorphic activity. Collectively, our data disclose the genetic and molecular etiology of a novel lethal syndrome with progeroid features, highlighting the importance of RTK signaling for human development and homeostasis.

Keywords: Xenopus; ASK1; ERK; RAF1; RASopathy.

Figure 1. RAF1 germline homozygous missense mutation causes a neonatal lethal syndrome with progeroid features

1. A

   Pedigree of the Turkish consanguineous family reporting the neonatal progeroid syndrome. Square, male; circle, female; black shading, affected individuals; small triangle, aborted; double lines, consanguineous marriages; diagonal line, deceased.

2. B

   Pictures of affected proband (III:2) at 50 days showing general progeroid features. Note subcutaneous lipoatrophy, severe hypotonia, distinctive craniofacial features with absence of external ears (insets), cleft hands and feet with oligodactyly.

3. C

   Schematic of the RAF1 protein structure showing the three conserved regions (CRs). p.T543M is located in the catalytic kinase domain (CR3).

4. D

   Multiple sequence alignment showing conservation of T543 across species and among RAF family members.

5. E

   RAF1^WT or RAF1^T543M were transiently overexpressed in HEK293T cells for 48 h, followed by 16 h of serum starvation and EGF stimulation for 15 min. Phos‐tag™ gel showing global levels of RAF1 phosphorylation and corresponding Western blot showing phosphorylation levels of Ser259 and Ser338 residues and total RAF1.

6. F, G

   All the RAF1 constructs were transiently overexpressed in HEK293T cells as described in (E). Western blotting showing (F) phosphorylated MEK1/2, total MEK1/2 and total RAF1 and (G) phosphorylated ERK1/2, total ERK1/2 and total RAF1.

Source data are available online for this figure.

Figure EV1. RAF1^T543M allele behaves as a loss‐of‐function mutation in vitro

1. A

   Genetic strategy. Combination of exome sequencing and homozygosity mapping. resulted in four candidate genes. Germline homozygous variants in PLIN2, ABL1, and C16orf93 were filtered out due to their low phylogenetic conservation and low predicted deleterious potential.

2. B

   Sanger sequencing chromatogram showing variant c.1628C>T (p.T543M) segregating with disease: homozygous in the proband (III:2), heterozygous in the parents (II:2 and II:3) and one sister (III:4) and absent in two other siblings (III:3 and III:6).

3. C

   Schematic representation of ERK1/2 MAP Kinase pathway.

4. D

   Quantification of phosphorylated ERK1/2, normalized to WT RAF1 in three independent Western blots (Error bars indicate mean ± SEM. Ordinary one‐way ANOVA).

Figure 2. RAF1^T543M allele behaves as a loss‐of‐function mutation in vivo

1. A

   Western blotting showing phosphorylated Erk1/2, total Erk1/2 and total Raf1 from Xenopus laevis embryos harvested at stage 28.

2. B

   Quantification of phosphorylated Erk1/2, normalized to WT Raf1 in three independent Western blots (Error bars indicate mean ± SEM. Ordinary one‐way ANOVA).

3. C

   Representative images illustrating the effect of WT or mutants RAF1 mRNA injection into Xenopus embryos, at stage 28.

4. D

   WISH performed on stage 10.5 Xenopus embryos injected with WT or mutants RAF1 mRNA. Xbra (blue staining) marking mesoderm induction. Vegetal (top), animal (middle) and lateral (bottom) views.

5. E

   WISH performed on stage 28 Xenopus embryos injected with WT or mutants RAF1 mRNA. Blue staining reveals Xbra or N‐tubulin markers depicting ectopic mesoderm induction, neural differentiation and brain posterization.

Source data are available online for this figure.

Figure 3. RAF1^T543M/T543M knock‐in cells reveal RAF1's compromised protein half‐life

1. A

   RAF1 homodimer (PDB:3OMV) showing the location of Thr543 (drawn as ball‐and‐stick) on helical subdomain. Active site is occupied by an inhibitor drawn as spheres.

2. B

   Positioning of Thr543 in the pocket.

3. C

   Met543 in the pocket. The Met543 side chain clashes despite being modeled in the least sterically hindered rotamer.

4. D

   Predicted side chain interaction of Thr543 and Tyr539 variants.

5. E

   Western blot downstream signaling potential of structural mutants that induce steric (p.T543M) or electrostatic (p.Y539E or p.T543D) interference.

6. F

   RAF1^T543M and RAF1^KO/KO HEK293T cells were generated by CRISPR‐Cas9. Top—bottom: Sanger‐sequencing of RAF1 in WT HEK293T cells; a RAF1^T543M clone showing the desired c.1628C>T (p.T543M) mutation and a synonymous PAM‐disrupting c.1632G>C; a RAF1^KO/KO clone with a homozygous 17 bp deletion.

7. G

   Confirmation of a clonal RAF1^T543M line by restriction‐digest of RAF1 exons 15–16 from gDNA of the genotypes shown with BccI. c.1628C>T introduces a BccI restriction site. The RAF1^WT/T543M heterozygote genotype was obtained from the patient's father.

8. H

   Western Blot for endogenous RAF1 in HEK293T cell lines of genotypes indicated, at steady state.

9. I

   Western‐blot showing that 8 and 24 h of epoxomicin treatment is sufficient to partially rescue RAF1 in RAF1^T543M/T543M cells. ACTIN serves as loading control.

Source data are available online for this figure.

Figure EV2. Loss‐of‐function mutation p.L603P impairs RAF1 kinase activity

Position of Leu603 and Thr543 in the RAF1 homodimer (PDB:3OMV). The p.L603P mutation causes an impairment of kinase activity, likely through destabilizing the αH helix and the helical bundle made by the αC and αH helices, and may also affect the dimer stability. Thr543 is located in the αE helix.

Figure 4. RAF1^T543M/T543M knock‐in cells are more sensitive to stress‐induced apoptosis

1. A

   Representative immunofluorescence images of 0.2, 0.8, and 1.6 mM H₂O₂ treated RAF1^T543M/T543M and RAF1^WT/WT HEK293T cells. Green: SYTOX dead cell stain. Blue: DAPI nuclei stain. Scale bar, 100 μm.

2. B

   Dose‐dependent SYTOX signal of RAF1^T543M/T543M and RAF1^WT/WT HEK293T cells incubated with increasing H₂O₂ concentrations for 2 h (n = 8 from two independent experiments, error bars indicate mean ± SEM. Two‐tailed Student's t‐test).

3. C

   PC tagged RAF1^WT or RAF1^T543M were transiently overexpressed along with ASK1 in HEK293T cells for 48 h, immunoprecipitated and blotted for ASK1. Ratio of ASK1 to RAF1 from the pulldown (PD) was quantified in three independent Western blots (Error bars indicate mean ± SEM. Ordinary one‐way ANOVA).

4. D

   Western blot documenting endogenous pASK1, pJNK levels and cleaved Caspase 3 following H₂O₂‐induced stress in isogenic HEK293T cells expressing endogenous RAF1^T543M/T543M or RAF1^WT. Note that pASK1 is increased without H₂O₂ treatment in RAF1^T543M/T543M compared with RAF1^WT.

5. E

   Proposed model of the negative impact of RAF1^T543M/T543M on proliferation owing to impaired MAPK/ERK transduction and increased apoptosis due to de‐repression of ASK1 signaling.

Source data are available online for this figure.

Figure EV3. Quantification of pASK1/ASK1 ratio in RAF1^WT and RAF1^T543M/T543M knock‐in cells

Quantification of phosphorylated ASK1, normalized to total ASK1 in three independent western blots (Error bars indicate mean ± SEM. Ordinary one‐way ANOVA).