PLX7904

Vemurafenib and dabrafenib block MEK-ERK1/2 signaling and cause tumor regression in the majority of advanced-stage BRAF(V600E) melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next-generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF(V600E) melanoma cells without paradoxical effects in wild-type cells. Furthermore, PLX7904 blocks the growth of vemurafenib-resistant BRAF(V600E) cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF(V600E) splice variant-mediated vemurafenib-resistant cells. We show that paradox-breaker RAF inhibitors potently block MEK-ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720-resistant cells harboring distinct BRAF(V600E) splice variants. These data support the further investigation of paradox-breaker RAF inhibitors as a second-line treatment option for patients failing on vemurafenib or dabrafenib.

BRAF inhibitors (BRAFi) are standard of care for the treatment of BRAF V600 mutation-driven metastatic melanoma, but can lead to paradoxical activation of the mitogen-activated protein kinase (MAPK) signalling pathway. This can result in the promotion of precancerous lesions and secondary neoplasms, mainly (but not exclusively) associated with pre-existing mutations in RAS genes. We previously reported a patient with synchronous BRAF-mutated metastatic melanoma and BRAF ;wt; /KRAS ;G12D;-metastatic colorectal cancer (CRC), whose CRC relapsed and progressed when treated with the BRAF inhibitor dabrafenib (GSK2118436). We used tissue from the resected CRC metastasis to derive a cell line, LM-COL-1, which directly and reliably mimicked the clinical scenario including paradoxical activation of the MAPK signalling pathway resulting in increased cell proliferation upon dabrafenib treatment. Novel BRAF inhibitors (PLX8394 and PLX7904), dubbed as "paradox breakers", were developed to inhibit V600 mutated oncogenic BRAF without causing paradoxical MAPK pathway activation. In this study we used our LM-COL-1 model alongside multiple other CRC cell lines with varying mutational backgrounds to demonstrate and confirm that the paradox breaker PLX8394 retains on-target inhibition of mutated BRAF V600 without paradoxically promoting MAPK signalling.

The recent studies have revealed that most BRAF inhibitors can paradoxically induce kinase activation by promoting dimerization and enzyme transactivation. Despite rapidly growing number of structural and functional studies about the BRAF dimer complexes, the molecular basis of paradoxical activation phenomenon is poorly understood and remains largely hypothetical. In this work, we have explored the relationships between inhibitor binding, protein dynamics and allosteric signaling in the BRAF dimers using a network-centric approach. Using this theoretical framework, we have combined molecular dynamics simulations with coevolutionary analysis and modeling of the residue interaction networks to determine molecular determinants of paradoxical activation. We have investigated functional effects produced by paradox inducer inhibitors PLX4720, Dabrafenib, Vemurafenib and a paradox breaker inhibitor PLX7904. Functional dynamics and binding free energy analyses of the BRAF dimer complexes have suggested that negative cooperativity effect and dimer-promoting potential of the inhibitors could be important drivers of paradoxical activation. We have introduced a protein structure network model in which coevolutionary residue dependencies and dynamic maps of residue correlations are integrated in the construction and analysis of the residue interaction networks.