Summary: type II RAF inhibition and relevance to BRAF fusion-driven pLGG

Tovorafenib is an oral, CNS-penetrant type II RAF kinase inhibitor of BRAF V600E, wild-type BRAF, and wild-type CRAF, with in vitro IC50 values of 7.1, 10.1, and 0.7 nM, respectively. Type I BRAF inhibitors such as dabrafenib act on monomeric BRAF V600E, do not block signaling from the dimer-dependent KIAA1549:BRAF fusion, and can cause paradoxical MAPK pathway activation in BRAF fusion-driven or BRAF wild-type tumors. Tovorafenib binds RAF monomers and dimers; preclinical studies in pediatric low-grade astrocytoma models showed equipotent activity against BRAF V600E and KIAA1549:BRAF with brain penetrance, and the EMA assessment found no paradoxical activation in BRAF fusion models. Tovorafenib is relatively sparing of ARAF, and in NF1 loss-of-function models without a BRAF alteration it increased ERK phosphorylation at low concentrations, which underlies the NF1-associated tumor warning. The mechanistic data apply to both the approved relapsed or refractory indication and the investigational first-line setting (FIREFLY-2).

Labeled mechanism of action

“Tovorafenib is a Type II RAF kinase inhibitor of mutant BRAF V600E, wild-type BRAF, and wild-type CRAF kinases.” (opens the source at this quote in a new tab)
“Tovorafenib exhibited antitumor activity in cultured cells and xenograft tumor models harboring BRAF V600E and V600D mutations, and in a xenograft model harboring a BRAF fusion.” (opens the source at this quote in a new tab)

“Tovorafenib is a central nervous system (CNS) penetrant, selective small molecule Type II RAF kinase inhibitor of mutant BRAF V600E, wild-type BRAF, and wild-type CRAF kinases, including RAF monomers and dimers and BRAF fusion, suppressing activation of the mitogen-activated protein kinase (MAPK) pathway” (opens the source at this quote in a new tab)

Binding mode and kinase potency

“Tovorafenib is a type-II RAF kinase inhibitor that inhibits RAF monomers and dimers.” (opens the source at this quote in a new tab)
“This binding mode is similar to that of sorafenib and differs from that of the Type-I BRAF inhibitor vemurafenib. In vitro, tovorafenib inhibited the kinase activity of BRAF V600E, WT BRAF, and WT CRAF kinases, with IC50 values of 7.1, 10.1, and 0.7 nM, respectively.” (opens the source at this quote in a new tab)

Rationale for a type II inhibitor in KIAA1549:BRAF fusion tumors

“These drugs (type I antagonists that target the "DFG-in" conformation of the kinase) fail to block signaling via KIAA1549:BRAF, a truncation/fusion BRAF oncoprotein which functions as a dimer and is found in the most common form of PLGA.” (opens the source at this quote in a new tab)
“We identify a type II RAF inhibitor that serves as an equipotent antagonist of BRAFV600E, KIAA1549:BRAF, and other noncanonical BRAF oncoproteins that function as dimers. This drug (MLN2480, also known as TAK-580) has good brain penetrance and is active on authentic human PLGA cells in brain organotypic cultures.” (opens the source at this quote in a new tab)

“Type I RAF inhibitors such as dabrafenib are not indicated for the treatment of tumors harboring a BRAF fusion due to paradoxical activation which may increase the risk of tumor growth” (opens the source at this quote in a new tab)

Paradoxical MAPK activation: BRAF fusion models versus NF1 loss-of-function models

“Data generated in preclinical BRAF fusion models suggest that tovorafenib is capable of inhibiting MAPK pathway signalling without inducing paradoxical activation in BRAF fusion–driven systems. However, the translational relevance of these models is limited, as they do not adequately reflect the genetic and biological characteristics of paediatric low-grade gliomas, particularly with regard to TP53 status.” (opens the source at this quote in a new tab)
“This effect is suggestive of paradoxical activation of the pathway in NF1-LOF tumour cell lines. Indeed, paradoxical activation by tovorafenib at subtherapeutic doses (0.01–0.1 µM) was also described in literature. This could be linked to tovorafenib being ARAF sparing.” (opens the source at this quote in a new tab)

RAF isoform selectivity: relative ARAF sparing

“We find that tovorafenib and naporafenib are most potent against CRAF but markedly less potent against ARAF.” (opens the source at this quote in a new tab)
“Type II RAF inhibitors are generally regarded as pan-RAF inhibitors, but our studies of these two agents, together with recent work with type II inhibitors belvarafenib and naporafenib, indicate that relative sparing of ARAF may be a property of multiple drugs of this class.” (opens the source at this quote in a new tab)

Tovorafenib (DAY101)

Mechanism of action