RAF inhibitor PLX8394 selectively disrupts BRAF dimers and RAS-independent BRAF-mutant-driven signaling

Abstract

Activating BRAF mutants and fusions signal as RAS-independent constitutively active dimers with the exception of BRAF V600 mutant alleles which can function as active monomers 1 . Current RAF inhibitors are monomer selective, they potently inhibit BRAF V600 monomers but their inhibition of RAF dimers is limited by induction of negative cooperativity when bound to one site in the dimer 1 – 3 . Moreover, acquired resistance to these drugs is usually due to molecular lesions that cause V600 mutants to dimerize 4 – 8 . We show here that PLX8394, a new RAF inhibitor 9 , inhibits ERK signaling by specifically disrupting BRAF-containing dimers, including BRAF homodimers and BRAF-CRAF heterodimers, but not CRAF homodimers or ARAF-containing dimers. Differences in the amino acid residues in the N-terminal portion of the kinase domain of RAF isoforms are responsible for this differential vulnerability. As a BRAF-specific dimer breaker, PLX8394 selectively inhibits ERK signaling in tumors driven by dimeric BRAF mutants, including BRAF fusions and splice variants and as well BRAF V600 monomers, but spares RAF function in normal cells in which CRAF homodimers can drive signaling. Our work suggests that drugs with these properties will be safe and useful for treating tumors driven by activating BRAF mutants or fusions.

Targeting Alterations in the RAF–MEK Pathway

“…© 2019 American Association for Cancer cancerdiscovery.aacrjournals.org Downloaded from REVIEW and these drugs (e.g., PLX8394), which are often referred to as "paradox breakers," are currently in phase I studies. These drugs inhibit signaling by specifically disrupting BRAFcontaining dimers, including BRAF-BRAF homodimers and BRAF heterodimers (43). These drugs are not effective against CRAF homodimers or ARAF-containing dimers likely due to amino acid residues situated at the N-terminus of the RAF kinase domain that leads to enhanced stabilization of the dimer interface in these dimers.…”

“…One alternative clinical strategy to target class II mutations involves the use of newer RAF inhibitors that function as RAF dimer inhibitors or RAF dimer breakers (17,43). These inhibitors can disrupt signaling from active BRAF dimers and can suppress MAPK signaling in these models.…”

“…In fact, RAF monomer inhibitors can induce paradoxical activation of BRAF and CRAF fusion constructs, likely through transactivation of the dimer partner, similar to what is observed with wild-type RAF dimers in the presence of activated RAS. " Paradox-breaker" RAF inhibitors, such as PLX8394, do not lead to paradoxical activation of BRAF fusions and can inhibit kinase activity-likely through the disruption of BRAF-containing dimers (43,70). However, PLX8394 cannot inhibit signaling from CRAF fusion dimers and actually leads to paradoxical activation of CRAF signaling in this setting (31).…”

The sixth international RASopathies symposium: Precision medicine—From promise to practice

“…A second class selectively disrupts BRAF containing homo and heterodimers, but spares CRAF homodimers (Yao et al, 2019). As predicted, these drugs inhibit Class I, II, and fusion driven tumors without obvious toxicity in patients.…”

“…Pan‐RAF inhibitors inhibit all tumors driven by BRAF mutant monomers, Class II BRAF mutants and fusions more potently than normal cells and RAS‐driven tumors. A second class selectively disrupts BRAF containing homo and heterodimers, but spares CRAF homodimers (Yao et al, ). As predicted, these drugs inhibit Class I, II, and fusion driven tumors without obvious toxicity in patients.…”