Mutually exclusive recurrent somatic mutations in MAP2K1 and BRAF support a central role for ERK activation in LCH pathogenesis

Abstract

Langerhans cell histiocytosis (LCH) is a myeloproliferative disorder characterized by lesions composed of pathological CD207(+) dendritic cells with an inflammatory infiltrate. BRAFV600E remains the only recurrent mutation reported in LCH. In order to evaluate the spectrum of somatic mutations in LCH, whole exome sequencing was performed on matched LCH and normal tissue samples obtained from 41 patients. Lesions from other histiocytic disorders, juvenile xanthogranuloma, Erdheim-Chester disease, and Rosai-Dorfman disease were also evaluated. All of the lesions from histiocytic disorders were characterized by an extremely low overall rate of somatic mutations. Notably, 33% (7/21) of LCH cases with wild-type BRAF and none (0/20) with BRAFV600E harbored somatic mutations in MAP2K1 (6 in-frame deletions and 1 missense mutation) that induced extracellular signal-regulated kinase (ERK) phosphorylation in vitro. Single cases of somatic mutations of the mitogen-activated protein kinase (MAPK) pathway genes ARAF and ERBB3 were also detected. The ability of MAPK pathway inhibitors to suppress MAPK kinase and ERK phosphorylation in cell culture and primary tumor models was dependent on the specific LCH mutation. The findings of this study support a model in which ERK activation is a universal end point in LCH arising from pathological activation of upstream signaling proteins.

Figure 1

Key genetic alterations identified in MAPK pathway genes in LCH patients.

Figure 2

MAPK pathway mutations identified in LCH patients. (A) Location of somatic MAP2K1 mutations identified in LCH patients in this study (top) and reported in other malignancies in the COSMIC database and recent literature (bottom). Also depicted in black are germ-line mutations observed in cardio-facio-cutaneous (CFC) syndrome. Triangles and bars indicate missense and in-frame deletion mutations, respectively. Figure not to scale. (B) Somatic mutations identified in multiple MAPK pathway members in LCH patients. C, carboxyl; D, docking; Mg, magnesium positioning loop; N, peptidyl; NES, nuclear export signal; NRR, negative regulatory region; P, phosphate binding; Pro, proline.

Figure 3

Functional characterization of the MAP2K1 mutants. (A) HEK293 cells were transiently transfected with expression plasmids encoding the indicated MAP2K1 and BRAF wild-type and mutant cDNAs, and corresponding lysates from cells maintained in serum were subjected to immunoblotting with the indicated antibodies. (B) Identification of functional implication of MAP2K1 mutations by IFC. Single cell suspension of LCH lesions were stained with Brilliant Violet (BV) 421-conjugated CD3, phycoerythrin (PE)-CD207, Alexa Fluor (AF) 488-phospho-MEK1, and AF647-phospho-ERK1/2 antibodies and analyzed by the gating strategy described in “Methods.” The top panels depict the distinctive CD3⁺ and CD207⁺ population sequestration (left) and overall p-MEK1⁺p-ERK1/2⁺ CD207⁺ (middle) or CD3⁺ (right) cells obtained by gating on 10 000 events. The middle and bottom panels show the representative images of random single cell events of 10 (138 through 2061) or 6 (308 through 4600) in CD207⁺ and CD3⁺ cells, respectively, identified by gating events with intermediate bright field (BF) area and BF aspect ratios close to 1. The indicated populations were then analyzed for coexpression of p-ERK1/2 or p-MEK1 in the CD207⁺ or CD3⁺ cells in the indicated channels. (C) Median fluorescent intensity (MFI) of p-MEK1 or p-ERK1/2 in CD207⁺ cells determined through IFC in patients harboring BRAFV600E (patients LCH5, LCH7, and LCH13) and MAP2K1 c.302_307del (patient LCH24) mutations, and patients with no somatic mutations (LCH30, LCH32, LCH37, LCH38, and LCH40).

Figure 4

Specific LCH mutations have differential sensitivity to BRAF and MEK inhibitors. (A,B) HE293 cells transiently transfected with expression plasmids encoding various cDNAs (A) or c.302_307del MAP2K1 lesion biopsy cell suspension (B) were treated with BRAF or MEK inhibitor for 1 hour, and corresponding lysates were subjected to immunoblotting with the indicated antibodies. (C) Median fluorescent intensity (MFI) of p-ERK1/2 in CD207⁺ cells determined through IFC analyses in patients harboring BRAFV600E (patients LCH5, LCH7, and LCH13) and MAP2K1 c.302_307del (patient LCH24) mutations, and patients with no somatic mutations (LCH30, LCH32, LCH37, LCH38, and LCH40) posttreatment with BRAF or MEK inhibitor.

Figure 5

OS and disease progression of LCH patients in this series by tumor genotype. (A) Kaplan-Meier analysis of overall survival stratified by presence of BRAFV600E, MAP2K1 mutation, or neither mutation (P < .66). (B) Percentage with progression or recurrence: MAP2K1-wt vs MAP2K1-mut, P < .1267 (i); BRAFV600E vs MAP2K1-mut, P < .0360 (ii); and BRAF-wt/MAP2K1-wt vs MAP2K1-mut, P < .4837 (iii).