DISCUSSION
The RAS-RAF-MEK-ERK cascade (RAS-RAF mitogen-activated protein kinase; MAPK) is the key regulatory pathway for cell growth, proliferation, differentiation, and apoptosis. Signaling through this pathway typically occurs through different plasma membrane growth factor receptors that trigger RAS family GTPases, including HRAS, NRAS and KRAS. Activated RAS proteins can recruit members of the RAF kinase family (ARAF, BRAF, and CRAF) to the plasma membrane, complex with and activate them. Activated RAF kinases signal downstream to other MEK/ERK cascade elements to over 150 downstream targets, nuclear and cytosolic. The MAPK pathway is frequently dysregulated in cancer, often via mutations of its intracellular components or activation of growth factor receptor tyrosine kinases. BRAF is the most potent form of RAF kinases13. T1799A transversion mutation in BRAF (BRAFv600) accounts for more than 80% of all known BRAF mutations and leads to hyperactivation of the MAPK pathway12,14 that results in deregulated downstream signaling and consequently, unregulated cell proliferation and survival, which contributes to oncogenesis. Patients harboring BRAF v600 mutations are treated by BRAF inhibitors, such as vemurafenib, dabrafenib and encorafenib. However, other BRAF mutations like Class III BRAF mutations were recently identified as being resistant to these agents.
In our patient, we identified two somatic mutations in the RAS pathway genes (BRAF N581I and HRAS G13V), which are mutually exclusive15. BRAF N581I is a Class III hotspot mutation that lies within the protein kinase domain of the Braf protein (UniProt.org). Class III BRAF mutants display low kinase activity or are kinase-dead. They require the co-existence of other upstream mutations to give the oncogenic signaling. They trigger the MAPK pathway through enhanced RAS binding and subsequent RAS-dependent CRAF activation resulting in elevated ERK signaling and are susceptible to ERK-mediated feedback16–18. (Supplementary Figure 4).
One study reported the cooperation of the BRAF and HRAS in histiocytic sarcoma. In this study, the BRAF mutation Phe595Leu (class III) co-occurred with HRAS (Q61R). The co-occurrence of these two mutations causes a higher oncogenic signal than in the case of BRAF alone19. This study also highlighted the efficacy of Pan-RAF inhibitors, such as sorafenib and AZ628, and the MEK inhibitor trametinib in interfering with the MEK/ERK phosphorylation driven by the cooperative activity of BRAF (F595L) and oncogenic RAS. Another study introduced the KIN-2787 as a potent and selective small-molecule pan-RAF inhibitor specifically designed to inhibit Class II and III BRAF dimers18.
Class III mutations are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. A multicenter analysis suggested that anti-EGFR therapy has promising effects for some patients with metastatic colorectal cancer that is not v600 BRAF mutated. The study demonstrated that half of the patients with kinase-impaired and RAS-dependent BRAF mutations responded to the therapy, whereas almost none of those with kinase-activating and RAS-independent mutations did20. In contrast, current RAF inhibitors are not expected to effectively inhibit the ERK pathway in these tumors. It was reported that RAF inhibitor vemurafenib failed to inhibit ERK signaling in tumor cells that express class III BRAF mutants. However, cell lines harboring Class III mutations are sensitive to classic RAS/MAPK singling inhibitors 21.