Abstract
Abstract
Peripheral T-cell lymphomas (PTCLs) are a heterogeneous and poorly understood group of aggressive non Hodgkin lymphomas with poor prognosis. To gain further insight on the genetics and pathogenic mechanisms of aggressive PTCLs we performed whole exome sequencing of matched tumor and normal DNA samples from 12 PTCL patients including 6 PTCL not otherwise specified (PTCL-NOS) tumors, 3 angioimunoablastic (AITL) T-cell lymphomas, 2 nasal type NK-/T-cell lymphomas and one enteropathy-associated T-cell lymphoma (EATL). This analysis identified 288 candidate coding somatic mutations in 268 genes and a mean mutation load of 24 non synonymous mutations per sample (range 4 – 57). Among these we noted the presence of a recurrent heterozygous mutation in the RHOA small GTPase gene (RHOA G17V) present in two independent AITL samples and one PTCL NOS biopsy. Analysis of a broad and diverse panel of 126 PTCL samples identified the presence of the RHOA G17V allele in 32 samples with a high prevalence in AITL (24/36, 67%, P < 0.001) and PTCL NOS cases (8/44, 18%, P < 0.002). The RHOA protein belongs to the Rho family of small GTPases, a group of Ras-like proteins responsible for linking a variety of cell-surface receptors to different intracellular signaling proteins. As is the case for RAS and most other small GTPases, RHOA activation is mediated by guanine exchange factors (GEFs), which catalyze the switch of RHOA from an inactive GDP-bound to an active GTP-bound state. Thus, and to test the functional significance of the RHOA G17V mutation we analyzed the capacity of this mutant to load GTP. This analysis revealed that RHOA G17V fails to incorporate GTP in response to an activated GEF in vitro. Moreover, and consistent with its inability to bind GTP, RHOA G17V failed to interact with rhotekin, a RHOA effector protein that selectively interacts with the GTP-bound active form of RHOA. However and most notably, the lack of RHOA G17V activation is not the result of a defect in RHOA-GEF interaction as RHOA G17V pull down assays demonstrated effective binding of this mutant protein to activated GEF proteins in T-cells. Based on these results we proposed an inhibitory role for RHOA G17V via sequestration of active GEF proteins. Consistently, while forced activation of RHOA signaling by GFP-RHOA overexpression induced loss of adhesion and round cell morphology in HEK293T cells, transfection of GFP-RHOA-G17V induced increased elongation and cellular protrusions as result of RHOA inactivation. In addition, immunoflourescence analysis of actin stress fiber formation by RHOA demonstrated effective abrogation of RHOA mediated cytoskeleton remodeling in cells expressing RHOA G17V. Overall these results show novel insight on the genetic basis of PTCLs and demonstrate a prominent role for RHOA G17V in the pathogenesis of AITL via disruption of RHOA signaling.
Disclosures:
No relevant conflicts of interest to declare.
