Abstract C153: Rare BRAF mutations in menlanoma and beyond: Rationalizing the afficacy of B-raf inhibitors via HPC-based in silico/in vitro investigations

Abstract

Abstract Melanoma is a highly aggressive type of cancer that affects the integumentary system and for which a family history of the disease, a fair complexion, the presence of a large number of moles, and skin exposure to natural/artificial ultraviolet (UV) radiation constitute established risk factors for malignancy development. The v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine protein kinase that plays a critical role in the RAS-RAF-MEK-ERK mitogen–activated protein kinase (MAPK) cell signaling pathway. Despite over 20 years of research into the regulation and function of the RAF proteins, it was only relatively recently that the BRAF isoform is mutated at a high frequency in different human cancers, identifying this kinase as another important oncogene along this pathway. FFocusing on melanoma, activating BRAF mutations were identified in 37%−53% of patients with the malignant form of this pathology, the most prevalent, missense kinase alteration being the replacement of the small, apolar residue valine (V) with the negatively-charged, phosphorylation mimicking glutamic acid (E) at position 600 (BRAFV600E) within the protein catalytic (i.e., ATP binding) site. It is interesting to note that BRAFV600E is not a UV-driven mutation; in fact, it may also be found in the molecular profile of atypical benign nevi; moreover, although BRAFV600E is the most frequent mutation (accounting for nearly 40% of all melanomas and in approximately 90% of BRAF-mutant melanomas), other more or less frequent activating variants in BRAF have also been reported, which include other aminoacidic substitutions at the same position (e.g., V600K/D/R), G469A/V/S, L597Q/R/S/V, A598V, and K601E/N/T, among others. Importantly, these mutations are also found in other cancers, e.g., thyroid cancer, colorectal cancer, and some brain malignancies including glioblastoma, pilocytic astrocytoma, and pediatric low-grade glioma. In this multidisciplinary work, we focused on some of these rare BRAF variants and studied their ability to interact with several, first-line FDA approved BRAF initbitors (BRAFis) from a structural, chemico-phisical and in vitro perspectives. To the purpose, we employed a combitation of in silico techniques, based on the use of high-performance computing (HPC), chemico-physical and spectroscopical techniques, and in vitro tests to verify the interactions between these mutated BRAF isoforms with the inhibitors. As a main point, we were able to determine the binding thermodinamics and kinetics of the selected BRAFis to the mutant BRAF proteins, and each BRAF/drug complex was characterized from a structural standpoint. All these information ultimately revelaed that some of the BRAF isoforms are able to effectively bind the corresponding BRAi, with consequent switch-off of the signaling patwhay, at least in vitro. The presented mutlidisciplinary protocol could be adoped in the future to predict the response of newly discovered BARF variants and/or variants of unknown significance towards current or newly-designed BRAFis. Citation Format: Sabrina Pricl, Erik Laurini, Domenico Marson, Gabriele Cavalieri. Rare BRAF mutations in menlanoma and beyond: Rationalizing the afficacy of B-raf inhibitors via HPC-based in silico/in vitro investigations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C153.