RHOAG17V Mutation Exhibits Markedly Different Functions in the Presence of TET2 Loss in T-Cells

Introduction: Angioimmunoblastic T cell lymphoma (AITL) is one of the most common types of PTCL, accounting for approximately 20% of cases. Genome sequencing analysis showed a recurrent RHOA^G17V mutation in up to 70% of AITL cases that frequently co-occurs with loss of function mutations in TET2.

RHOA controls a wide range of biological functions, including cell adhesion, migration, proliferation, survival, and cytoskeleton remodeling. When the RHOA residue 17 glycine is replaced with valine, the GTP binding pocket is closed, resulting in blocked GTP_GDP interconversion and it is predicted to function in a dominant negative role¹. How RHOA^G17V contributes to the pathogenesis of AITL is not entirely clear and how RHOA^G17V interacts with TET2_KO has not been elucidated.

Methods: We used the CRISPR-Cas9 system to generate TET2_KO, knock-in of RHOA^G17 and double TET2 KO/RHOA^G17V mutants (DM) in primary human CD4+ T-cells isolated from PBMCs of healthy donors and performed cellular proliferation and functional analyses of the mutant and wild-type (WT) cells. We also performed imaging analysis to compare changes in actin polymerization and immunological synapse (IS) formation in the different groups.

Results: The cell growth kinetics and proliferation of RHOA^G17V mutant were similar to the WT but they were significantly increased in TET2_KO and DM cells. RHOA is crucial for the organization of the actin cytoskeleton. We, therefore, examined actin polymerization and distribution during the proliferation cycle induced by CD3/28 beads stimulation. Surprisingly, actin polymerization was persistently increased and maintained a membranous distribution in TET2_KO and DM cells in comparison to RHOA^G17V and WT². Because IS plays an important role in T-cell activation and function, we also imaged IS between T-cells and staphylococcus enterotoxin (SE) pulsed Raji cells and found significantly higher IS intensity in TET2_KO and DM cells compared to RHOA^G17V and WT cells. We also assayed for ROCK kinase and RAC1 activation in the modified cells after stimulation with CD3/28 beads for 5 and 30 min and found significantly higher RAC1 activity in unstimulated TET2_KO and DM cells. However, with 30 min stimulation, it decreased significantly compared to WT cells. In contrast, ROCK kinase showed the opposite pattern to RAC1. These profiles could not be simply explained by RHOA^G17V acting just in a dominant negative role.

Since there is a strong association between TET2_LOF mutation, RHOA^G17V mutation, and Tfh lymphomas. We examined the expression of a number of Tfh and memory cell markers. We found that ICOS, CCR7, PD1, CD45RO, and BCL6 were increased in all modified cells in comparison to WT but more markedly in DM cells¹. Because TET2 plays an important role in catalyzing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) we found significantly increased 5mC and decreased 5hmC in TET2_KO cells but DM cells surprisingly showed even more significant changes.

We also examined the proximal TCR signaling pathway activation after stimulation with CD3/28 beads for 5 and 30 min. By Western Blotting, we found significantly increased activation of p-AKT(Ser473), and NF-kB p65 (Ser536) in DM cells at 30 minutes of stimulation, whereas P-Foxo1(Ser256) was significantly decreased. P-mTOR (Ser2448), P-PLCg(y783), P-ERK1/2(Thr202/Tyr204) did not show significant changes.

Discussion: Using a knock-in approach to allow normal levels of expression of RHOA^G17V in human CD4 T-cells, we observed only minor functional variations from WT cells. In combination with TET2_KO, however, striking changes were observed. TET_KO reprograms many aspects of T-cell function and RHOA^G17V mutations built upon this altered functional state and mediated additional changes. Our study strongly supports the cooperation between RHOA^G17V mutation and TET2- loss but the mechanistic basis behind the cooperativity of these mutations requires further investigation. Overall, using normal human cells and a novel approach of knock-out and knock-in of specific mutants offers new insights into the role of TET2 and RHOA^G17V mutations in AITL pathogenesis and provides a model to further study the mechanisms of their interaction.

References:

1. Weinstock; et. al. Blood 2018; 132(9):935-947
2. Liu et. al. Protein Cell 2021, 12(7):578–585