Cooperative Activity of BRAF F595L and Mutant HRAS in Histiocytic Sarcoma Provides New Insights into Oncogenic BRAF Signaling
Authors: Kordes Maximilian , Röring Michael , Heining Christoph , Braun Sandra , Hutter Barbara , Richter Daniela , Geörg Christina , Scholl Claudia , Roth Wilfried , Rosenwald Andreas , Geissinger Eva , von Kalle Christof , Jäger Dirk , Brors Benedikt , Weichert Wilko , Grüllich Carsten , Glimm Hanno , Brummer Tilman , Fröhling Stefan 
Published: October 2019

Abstract
Activating BRAF mutations, in particular V600E/K, drive many cancers, including a substantial proportion of systemic histiocytic disorders, and mutant BRAF-selective inhibitors are promising therapeutics for these diseases. Activating BRAF alleles are considered mutually exclusive with mutations in RAS family members, whereas inactivating BRAF mutations in the D(594)F(595)G(596) motif can coexist with oncogenic RAS and cooperate via transactivation of wildtype RAF proteins and paradoxical MEK/ERK activation. Due to the increasing use of global approaches to tumor genomic profiling, many non-V600 BRAF mutations are being detected whose functional consequences and therapeutic actionability are often unknown. We used several in vitro experimental systems, including Braf-deficient murine embryonic fibroblasts expressing a regulatable HRAS oncogene, to determine the biochemical properties and cellular effects of a largely uncharacterized mutation, F595L, in the DFG motif of the BRAF activation segment that was identified by clinical exome sequencing in a patient with histiocytic sarcoma and multiorgan involvement and also occurs as somatic alteration in colorectal adenoma or carcinoma, non-small cell lung cancer, cholangiocarcinoma, urothelial cancer, melanoma, and neuroblastoma and as germline mutation in cardio-facio-cutaneous syndrome. In addition, we investigated the interaction between BRAF F595L and a concomitant HRAS Q61R allele, which was present in the same tumor cell clone and occurs as acquired alteration in multiple tumor types and as inherited variant in Costello syndrome. Unlike previously described DFG motif mutants, BRAF F595L is a gain-of-function variant with intermediate activity towards MEK that, in sharp contrast to BRAF V600E, requires an intact dimer interface for downstream signaling. Furthermore, BRAF F595L does not act paradoxically, but nevertheless cooperates with mutant HRAS to induce maximal activity of the MEK-ERK signaling pathway. Of immediate clinical relevance, BRAF F595L shows divergent responses to the mutant BRAF-selective inhibitors vemurafenib and dabrafenib, whereas signaling driven by BRAF F595L with and without mutant HRAS is efficiently blocked by the pan-RAF inhibitors sorafenib and AZ628 and the MEK inhibitor trametinib. Consistent with this, sorafenib treatment led to abrogation of aberrant MEK/ERK signaling in the index case with histiocytic sarcoma driven by BRAF F595L and HRAS Q61R. Mutation data from patients and cell lines, representing 18 different tumor entities, show that BRAF F595L as well as other BRAF mutants with intermediate signaling activity coincide with mutant RAS in at least 40% and 23% of cases, respectively. These data define a distinct class of activating BRAF mutations that cooperate with oncogenic RAS in a non-paradoxical fashion, extend the spectrum of patients with systemic histiocytoses and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway, and underscore the value of comprehensive genomic profiling for uncovering the vulnerabilities of individual tumors.

Disclosures
Off Label Use: Administration of sorafenib in a patient with histiocytic sarcoma.