Role and Mechanisms of Rhoa G17V in the Pathogenesis of AITL

Abstract

Peripheral T cell lymphomas (PTCL) are a heterogeneous group of lymphoid tumors resulting from the malignant transformation of mature T-cells. Among these, angioimmunoblastic T-cell lymphoma (AITL), which accounts for almost 20% of PTCL cases, represents one of the most aggressive forms of non-Hodgkin lymphoma with a dismal survival rate of 30% at 5 years. AITL tumors are characterized by loss of function mutations in the Ten-Eleven Translocation 2 (TET2) epigenetic tumor suppressor and a highly recurrent mutation (p.Gly17Val, G17V) in the RHOA small GTPase gene, present in almost 70% of AITL cases. Immunohistochemical analyses and gene expression profiling studies have established follicular T helper cells (Tfh), a as the cell of origin of AITL. Tfh cells are a CD4+ T-cell population characterized by high levels of expression of the chemokine receptor CXCR5, the inhibitory receptor PD-1 and the transcription factor BCL6. To investigate the specific role and mechanisms of RHOA G17V in AITL we first generated a conditional inducible Rhoa G17V knock-in mouse by introducing a floxed Rhoa wild type minigene upstream of the Rhoa G17V mutant exon in the endogenous RhoA locus by homologous recombination. RhoA G17V conditional knockin mice express wild type RhoA but switch to express the mutant G17V allele upon expression of Cre recombinase. Crossing this line with a CD4CreERT2 strain, which expresses a tamoxifen-inducible form of Cre, results in tamoxifen inducible selective activation of Rhoa G17V specifically in CD4 T-cells. Given the close association of the RHOA G17V mutation with AITL we hypothesized that expression of mutant Rhoa G17V could promote Tfh cell polarization and transformation, explaining the high prevalence of this mutation in AITL cases. Notably, tamoxifen-induced expression of Rhoa G17V in CD4CreERT2 RhoA G17V knockin mice resulted in spontaneous accumulation of Tfh cells expressing Cxcr5, Pd-1 and Bcl6 in absence of immunization. Mechanistically, RhoA G17V Tfh cells expressed increased levels of the ICOS receptor, a major factor in Tfh development, and showed enhanced ICOS signaling. Moreover, suppression of ICOS signaling with an anti-ICOSL blocking antibody impaired Rhoa G17V-induced Tfh polarization. Next, we analyzed the specific interaction of RHOA G17V and loss of Tet2 in AITL. Expression of mutant RHOA G17V in hematopoietic progenitors from Tet2 knockout mice resulted in the specific development of AITL with a median latency of 14 months. Tet2-/- RHOA G17V tumors are clonal and transplantable and present typical AITL Tfh-like features including expression of CXCR5, PD-1, BCL6 and ICOS, and are critically dependent on ICOS signaling for growth and proliferation in vivo. In all, our results demonstrate an instructive role for the RHOA G17Vmutation in Tfh cell fate specification and in AITL transformation and support an oncogenic role for ICOS signaling in the pathogenesis of AITL. The AITL animal model reported here may facilitate the preclinical testing of new therapeutic approaches for the treatment of this disease. DisclosuresNo relevant conflicts of interest to declare.