Alternative genetic mechanisms of BRAF activation in Langerhans cell histiocytosis

Abstract

Langerhans cell histiocytosis (LCH) is characterized by inflammatory lesions containing pathologic CD207⁺ dendritic cells with constitutively activated ERK. Mutually exclusive somatic mutations in MAPK pathway genes have been identified in ∼75% of LCH cases, including recurrent BRAF-V600E and MAP2K1 mutations. To elucidate mechanisms of ERK activation in the remaining 25% of patients, we performed whole-exome sequencing (WES, n = 6), targeted BRAF sequencing (n = 19), and/or whole-transcriptome sequencing (RNA-seq, n = 6) on 24 LCH patient samples lacking BRAF-V600E or MAP2K1 mutations. WES and BRAF sequencing identified in-frame BRAF deletions in the β3-αC loop in 6 lesions. RNA-seq revealed one case with an in-frame FAM73A-BRAF fusion lacking the BRAF autoinhibitory regulatory domain but retaining an intact kinase domain. High levels of phospho-ERK were detected in vitro in cells overexpressing either BRAF fusion or deletion constructs and ex vivo in CD207⁺ cells from lesions. ERK activation was resistant to BRAF-V600E inhibition, but responsive to both a second-generation BRAF inhibitor and a MEK inhibitor. These results support an emerging model of universal ERK-activating genetic alterations driving pathogenesis in LCH. A personalized approach in which patient-specific alterations are identified may be necessary to maximize benefit from targeted therapies for patients with LCH.

Figure 1.

Novel genomic alterations in BRAF in patients with LCH results in constitutive ERK activation, which is responsive to second-generation BRAF and MEK inhibitors but not to a BRAF-V600E inhibitor. (A) Illustration of the FAM73A-BRAF fusion identified in this study. (B) Illustration of the BRAF exon 12 in-frame deletions identified in this study. (C) HEK293 cells were transiently transfected with expression plasmids encoding the indicated BRAF wild-type, fusion, and mutant cDNAs, and corresponding lysates from cells maintained in serum were subjected to immunoblotting with the indicated antibodies. Where indicated, cells were treated with BRAF or MEK inhibitor for 4 hours before harvest. (D) LCH lesion biopsy cell suspension harboring indicated BRAF mutations or fusion were treated with BRAF or MEK inhibitor for 4 hours and corresponding lysates were subjected to immunoblotting with the indicated antibodies. (E) LCH lesion biopsy cell suspension harboring indicated BRAF mutations (BRAF-V600E or in-frame exon 12 deletions [indel]) or fusion (FAM73A-BRAF) were treated with BRAF or MEK inhibitor for 4 hours. Median fluorescent intensity (MFI) of p-ERK1/2 in CD207⁺ cells were determined through IFC analyses post treatment with various MAPK pathway inhibitors. (F) Pie chart showing the estimated distribution of genetic alterations identified in MAPK pathway genes in patients with LCH from an institutional cohort, including the novel BRAF fusion and deletions. The frequencies of MAPK pathway mutations were estimated based on previously reported data and the current study (supplemental Methods).^, The asterisk indicates single cases.