RAF Dimerisation and Paradoxical Activation by BRAF Inhibitors

RAF Works as a Dimer

The RAF kinases (ARAF, BRAF, CRAF) are activated at the membrane by RAS-GTP, which promotes them to pair into dimers. Within a dimer, one protomer allosterically switches on the other.

BRAF V600E is an exception: it is active as a monomer and does not need RAS or dimerisation, which is why it can be targeted cleanly in V600E tumours.

The Paradox

First-generation BRAF inhibitors bind one protomer of a RAF dimer and, through negative cooperativity, actually enhance the activity of the drug-free partner. In a cell with high RAS-GTP, such as a RAS-mutant cell, there are plenty of RAF dimers, so the net effect is increased ERK signalling.

Clinically this manifests as the development of cutaneous squamous cell carcinomas and keratoacanthomas in patients on single-agent BRAF inhibitors, driven by pre-existing RAS mutations in skin cells.

Consequences for Non-V600 and Fusion BRAF

Class 2 BRAF mutants and BRAF fusions signal as dimers, and class 3 mutants depend on CRAF dimers driven by upstream RAS. First-generation inhibitors are poorly effective, or paradoxically activating, against these.

This is a major reason BRAF alterations are stratified into classes, with different or no targeted options for the non-V600 groups.

Newer Inhibitor Designs

Adding a MEK inhibitor mitigates the paradox and reduces secondary skin cancers, which is now standard with BRAF-inhibitor therapy. Paradox-breaker and pan-RAF dimer inhibitors are designed to bind both protomers or to avoid inducing the active conformation.

These aim to extend RAF targeting to class 2 and 3 mutants and BRAF fusions, and several are in clinical development.

Interpretation Notes

A BRAF alteration report should specify the exact change; V600E behaves very differently from non-V600 mutations and fusions with respect to available drugs.

A RAS mutation is a reason not to use a single-agent first-generation BRAF inhibitor, because of paradoxical pathway activation.

Key Takeaways

• ·RAF kinases signal as dimers; BRAF V600E is an exception that works as a monomer.
• ·First-generation BRAF inhibitors transactivate the partner protomer, activating ERK in RAS-mutant cells.
• ·This causes secondary skin tumours and makes the drugs unsuitable for non-V600 BRAF.
• ·MEK-inhibitor combinations and paradox-breaker inhibitors address the problem.