Genetic basis for iMCD-TAFRO

Abstract

TAFRO syndrome, a clinical subtype of idiopathic multicentric Castleman disease (iMCD), consists of a constellation of symptoms/signs including thrombocytopenia, anasarca, fever, reticulin fibrosis/renal dysfunction, and organomegaly. The etiology of iMCD-TAFRO and the basis for cytokine hypersecretion commonly seen in iMCD-TAFRO patients has not been elucidated. Here, we identified a somatic MEK2^P128L mutation and a germline RUNX1^G60C mutation in two patients with iMCD-TAFRO, respectively. The MEK2^P128L mutation, which has been identified previously in solid tumor and histiocytosis patients, caused hyperactivated MAP kinase signaling, conferred IL-3 hypersensitivity and sensitized the cells to various MEK inhibitors. The RUNX1^G60C mutation abolished the transcriptional activity of wild-type RUNX1 and functioned as a dominant negative form of RUNX1, resulting in enhanced self-renewal activity in hematopoietic stem/progenitor cells. Interestingly, ERK was heavily activated in both patients, highlighting a potential role for activation of MAPK signaling in iMCD-TAFRO pathogenesis and a rationale for exploring inhibition of the MAPK pathway as a therapy for iMCD-TAFRO. Moreover, these data suggest that iMCD-TAFRO might share pathogenetic features with clonal inflammatory disorders bearing MEK and RUNX1 mutations such as histiocytoses and myeloid neoplasms.

Fig. 1. Clinical presentation and genetic features of patients with iMCD-TAFRO.

a Hematoxylin & eosin (H&E) and reticulin stain of a lymph node (LN) and bone marrow (BM) at diagnosis and after treatment (Patient 2; magnifications are indicated). b, c Representative plain CT (b) and PET scan (c) images of Patient 2 at diagnosis and after treatment. d, e Lollipop graphs showing frequencies of mutations in MEK2 (d) and RUNX1 (e) across cancers. Figures were made based on the cBioPortal data of 42,049 samples derived from TCGA PanCancer Atlas Studies and curated set of non-redundant studies (158 studies in total) and the location of mutations that were identified in Patient 1 and 2 are highlighted. Mutations affecting MEK2^P128 were identified in 3 bladder carcinoma, 1 melanoma, and 1 pancreatic ductal adenocarcinoma. f Structure of WT Runt homology domain (white) and CBFβ (gray) with RUNX1^G60C position highlighted in red. Figure was made based on PDB ID: 1E50.

Fig. 2. Pathogenic roles of mutant MEK2 and mutant RUNX1.

a, b Cell growth of isogenic 32D cells cultured with various concentration of mouse IL-3 (a). Heatmaps were made based on relative cell growth of MEK2^WT and MEK2^P128L expressing cells compared to EV (empty vector)-transduced cells in b (n = 3 per genotype; the mean percentage ± standard deviation (SD); two-way ANOVA with Dunnett’s multiple comparison test). c Representative western blot (WB) analysis of isogenic 32D cells from three biologically independent experiments with similar results. Experimental design is shown above. d Representative immunohistochemistry of phospho-ERK1/2 for the lymph nodes from both patient 1 and 2 as well as a normal tonsil as a control (original magnification x100). e Dose response curves of isogenic 32D cells to trametinib (n = 3; the mean value ± SD is shown). IC₅₀ value for each genotype is indicated. f Cell growth of isogenic 32D cells cultured with various concentration of trametinib. Heatmaps were made based on relative cell growth of MEK2^WT and MEK2^P128L expressing cells compared to EV (empty vector)-transduced cells in Supplementary Fig. 2c-e. g Representative WB analysis of isogenic 32D cells from three biologically independent experiments with similar results. h Luciferase activity of isogenic THP-1 cells retrovirally transduced with indicated constructs (top) and representative WB analysis of the same cells using an antibody against RUNX1 which recognizes N-terminus of RUNX1 protein (bottom). These cells were transfected with a M-CSF receptor reporter plasmid, and luciferase activities were measured as previously described [18] and presented as the fold change relative to EV-transduced cells (AE: RUNX1/RUNX1T1 (AML1/ETO); n = 3; the mean + SD; one-way ANOVA followed by Holm-Sidek’s multiple comparison test). i Luciferase activity of HEK293T cells transfected with the reporter plasmid and mammalian expression vectors for RUNX1^WT and RUNX1^G60C at various doses as indicated (data are shown as in h). j, k Results of serial replating assays using primary mouse BM cells transduced with indicated constructs (n = 3 per genotype). Number of colonies (j) (the mean ± SD; two-way ANOVA with Tukey’s multiple comparison test; p-values compared to EV-expressing cells are shown), and representative images of colonies (k) (original magnification x 20) are shown. *p < 0.05; **p < 0.01; ***p < 0.001.