Abstract LB-B10: Targeting a new allosteric site modulates selectivity of BRAF dimer inhibition
Authors: Agianian Paul (Bogos) , Cotto-Rios Xiomaris M , Gitego Nadege , Zachariouadakis Emmanouil , Gavathiotis Evripidis 
Published: March 2020

BRAF mutants are present in ~50% of melanoma patients as well as in colorectal (~10%), thyroid carcinomas (~40%) and other malignancies. FDA-approved RAF inhibitors Vemurafenib, Dabrafenib and Encorafenib show remarkable responses in melanoma patients with BRAFV600E tumors. However, these drugs ultimately become ineffective due to adaptive and acquired resistance mechanisms, many of which depend on signaling by poorly inhibited BRAF homo- or hetero-dimers. In about 20-30% of melanoma tumors, intrinsic resistance is driven by a constitutive BRAF dimer splice variant, p61-BRAFV600E, while in resistant colorectal and thyroid tumors RAF dimerization is maintained by frequent RAS activation. An adaptive resistance mechanism depends on inhibitor-induced relief of ERK-mediated feedback pathway inhibition, which stimulates RAS-GTP and increases RAF dimers. Finally, oncogenic non-V600E BRAF species in some tumors signal as obligated dimers. Challenged by the clinical relevance of BRAF dimerization, we initiated a systematic quest for novel inhibitors that specifically recognize BRAFV600E dimers. We developed a phenotypic cellular assay to screen for inhibitors of ERK-signaling, using VEM-resistant melanoma cells that express p61-BRAFV600E dimers. We discovered new potent BRAF inhibitors, including a FDA approved kinase inhibitor. We obtained novel crystal structures of this drug with BRAFV600E and BRAFWT. We used these structural insights and scaffold-based drug-design methods to develop PHI1, a first-in-class αC-IN BRAF inhibitor. Remarkably, the crystal structure of BRAFV600E/PHI1 complex revealed that this compound targets a new BRAF allosteric site, which uniquely modulates the αC-helix conformation. In vitro and cellular studies showed that PHI1 is potent and selective against resistant cells with p61-BRAFV600E dimers. In addition, SAR analysis indicated that selectivity for dimer inhibition is lost upon modification of PHI1’s moiety targeting the allosteric pocket, suggesting that this site modulates specificity for BRAF dimers. Co-treatment experiments revealed that PHI1 inhibits the second site within the dimers with positive cooperativity driven from first site occupancy by αC-OUT inhibitors, in contrast to current RAF inhibitors that exhibit no, or negative, cooperativity. Based on these data, co-treatment of PHI1 with various clinical RAF and MEK inhibitors was explored in BRAF dimer-dependent tumors resulting in promising synergistic effects. This work provides a new class of allosteric BRAF inhibitors that may broadly diminish clinically untargeted oncogenic signaling dependent on BRAF dimers.

Citation Format: Paul (Bogos) Agianian, Xiomaris M Cotto-Rios, Nadege Gitego, Emmanouil Zachariouadakis, Evripidis Gavathiotis. Targeting a new allosteric site modulates selectivity of BRAF dimer inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B10. doi:10.1158/1535-7163.TARG-19-LB-B10