Co-Occurrence of TP53/PTEN and CCR4 genetic Alteration Deregulates T_H-Cell Differentiation and Drives T_H2-like Lymphomagenesis and T-Cell Signaling

Background Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of post thymic T-cell neoplasms, with 40% not further classifiable categorized as PTCL, not otherwise specified (PTCL-NOS). Using gene expression profiling, we identified two novel molecular subgroups (i.e., PTCL-GATA3 and PTCL-TBX21) within PTCL-NOS cases designated with distinct T-helper (T_H) transcriptional programs and clinicopathological features. PTCL-GATA3 represents 40% of PTCL-NOS and is defined by its high expression of GATA3 and its transcriptional target genes. It is clinically more aggressive when compared to other PTCL entities and is characterized by co-deletion of PTEN and TP53. This suggests these genetic events are critical in PTCL-GATA3 pathogenesis and in T_H2-cell lymphomagenesis. The mutation spectrum revealed mutations in CCR4, a GATA3 transcriptional target, are found more frequent in PTCL-GATA3 than in PTCL-TBX21, with many of them located in the cytoplasmic domain and gain-of-function in nature. While earlier studies have focused more on how the cytoplasmic domain can regulate ligand specificities (CCL17 and CCL22) and hence, migration, we aim to define the potential role of CCR4 mutants in T_H2-cell differentiation and PTCL-GATA3 pathogenesis. Within, we delineated functional applications associated with these lesions within the PTCL-GATA3 subgroup.

Methods Clinical outcome data were assessed with the Kaplan-Meier method. Mutation data was generated from whole exome sequencing (n = 112). CD4-specific Cre, Trp53^R172H, and Pten deleted murine mouse models were developed on a C57BL/6 background. Chromatin immunoprecipitation- quantitative PCR (CHIP-qPCR) and luciferase assays were performed to elucidate the role of TP53 in GATA3 inhibition. Luciferase assays included co-transduction of TP53-expression vectors and luciferase vectors (pGL3-E) cloned with GATA3 regions of interest. In vitro analysis of ectopic expression of the CCR4 cytoplasmic domain mutants was conducted using healthy-donor CD3⁺ T-cells or the CD4⁺ Jurkat T-cell line. T-cell receptor signaling was determined by flow cytometry analysis of p-Lck, p-SLP76, and p-ZAP70. Protein-of-interest levels were assessed by Western blotting. Following corrections for false discoveries, p-values < 0.05 were considered significant.

Results Human PTCL-GATA3 cases had an inferior clinical outcome compared to other PTCL entities, especially when a co-deletion of TP53 and PTEN was present. These same findings were observed in murine models, suggesting that Trp53^mut provided accelerated, cooperative oncogenic impetus in the presence of Pten deletions. We observed an increase in T-cell receptor activation in Trp53^mut CD4⁺ murine T-cells, indicating TP53 plays a key role in regulating T-cell activation and signaling. Through CHIP-qPCR, TP53 showed binding affinity to many locations in GATA3 promoter regions and a region in intron 3. Further validation through luciferase assays revealed that increasing TP53 expression led to repression in luciferase activity at the intron 3 region, suggesting TP53 binding at this locus was inhibitory. In vitro analyses demonstrated that ectopic expression of CCR4 cytoplasmic domain mutants led to an observed increase T-cell receptor activation, AKT/mTOR signaling, and migration in Jurkat cells in comparison to control vectors, especially in CCR4 mutants that did not co-occur with TP53 mutations in clinical cases.

Conclusions Our findings establish important roles that mutations in TP53, PTEN, and CCR4 have in PTCL-GATA3 pathogenesis. The integrated sequencing and in vitro findings suggest TP53 was identified to have roles in T-cell differentiation and T-cell receptor signaling. TP53 is also shown to play a direct role in regulating GATA3, and thus T_H2 differentiation, expanding beyond its canonical role in cell cycle regulation. CCR4 cytoplasmic domain mutations (Q330X and Q349X), that do not co-occur in PTCL-GATA3 cases with TP53 mutations, led to an increase in TCR signaling. However, this same result was not observed in CCR4^Y331X,which is commonly associated with TP53 mutation in PTCL-GATA3 clinical cases, suggesting these mutations can increase in TCR signaling to enhance lymphomagenesis in the absence of mutant TP53. These findings indicate that the occurrence of these lesions activates divergent pathways, resulting in T-cell transformation towards the T_H2 phenotype.

No relevant conflicts of interest to declare.