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The RAS-MAPK pathway is a hotspot for somatic mutations in human cancer and for germline mutations in the RASopathy developmental syndromes. Understanding the regulatory mechanisms that govern this pathway is critical for identifying new drug targets and establishing effective therapeutic approaches for RASopathy and cancer patients alike. RASopathies are caused by mutations in many of the same genes as those found in cancer, however RASopathy mutations must be tolerated during development and as a result tend to be distinct those in cancer in terms of their location within the protein or in which family member or isoform they occur. In the case of the RAF kinase, BRAF, oncogenic mutations occur overwhelmingly in the catalytic (CAT) region, whereas RASopathy mutations are also frequently reported in cysteine-rich domain (CRD). The CRD is a zinc-stabilized lipid and protein binding domain in the RAF regulatory (REG) region. Previous studies have shown that the CRD plays roles in RAS activation of RAF by interacting with phosphatidylserine (PS) in the plasma membrane and by bindings directly to RAS GTPases. Conversely, the CRD also plays a role in the autoinhibitory interactions of the REG and CAT domains under quiescent conditions. Indeed, recently published cryo-EM structures of autoinhibited BRAF show the CRD bound to the CAT domain in cis and to the 14-3-3 dimer that stabilizes the REG-CAT interactions in trans. However, the relative importance of these functions and the residues which mediate them are not well understood. Through the characterization of a panel of RASopathy CRD mutations we show that they can be grouped into three distinct classes based on their abilities to relieve autoinhibition and/or enhance PS binding, with relief of autoinhibition being the major factor determining BRAF biological activity and mutational severity in zebrafish developmental studies. Encouragingly, these findings tracked well with RASopathy patient reports, indicating the utility of our approaches as prognostic indicators of RASopathy mutation severity in patients. Further, we found that the CRD is required to maintain BRAF in a non-signaling state and mutations which disrupt this function result in elevated RAS-dependent and RAS-independent BRAF function. Importantly, our studies indicate that stabilizing CRD-mediated autoinhibition may be a viable approach to prevent the aberrant BRAF activation found in many cancers and the RASopathy syndromes. Finally, when we compared the properties of BRAF CRD to those of CRAF, we found that that BRAF CRD is a stronger mediator of both autoinhibition and PS binding. In contrast, my preliminary studies indicate that CRAF CRD may instead play a greater role in direct RAS binding. Moreover, despite its reduced role in autoinhibition, CRAF CRD retains its ability to bind 14-3-3 proteins and we are currently investigating the interplay of 14-3-3, RAS and the CRD in regulating CRAF function. Citation Format: Russell Spencer-Smith, Elizabeth M. Terrell, Christine Insinna, Constance Agamasu, Morgan E. Wagner, Daniel A. Ritt, Jim Stauffer, Andrew G. Stephen, Deborah K. Morrison. Germline RASopathy mutations provide insights into the differential regulation of RAF family kinases [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR06.

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.