Characterization of RHOA pathway mutations in CTCL. (A) Distribution of RHOA mutations in malignancies of mature CD4⁺ T cells. There are no RHOA mutations in anaplastic large cell lymphoma. (B) Schematic of RHOA activation pathways. ARHGEF3 is a RHOA guanine nucleotide exchange factor that activates RHOA by catalyzing the replacement of GDP to GTP. (C) Identification of somatic mutations in ARHGEF3. The arginine at the 110th amino acid position is highly conserved across species. (D) Expression of WT ARHGEF3 (WT) or mutants (p.R110H; p.L301E) in HEK293T cells. We show immunoblot analysis of ARHGEF3 and loading control (β-actin) in lentivirally transduced HEK293T cells. (E) Effects of ARHGEF3 mutations on RHOA activity. HEK293T cells were transduced with WT ARHGEF3 or its mutants. RHOA-GTP levels were assessed by the G-LISA assay (Cytoskeleton). These data represent 6 biological replicates using repeat assays performed in 3 independently made lentivirally transduced cell lines. Each cell line was subject to 2 assays. The horizontal lines indicate the mean ± standard error from 6 independent biological replicates performed. P value was determined by 2-sided paired ratio t test. *P < .05.