Liu, Xuxiang

Peripheral T cell lymphoma (PTCL) is a heterogeneous group of postthymic T cell neoplasms, with ~40% classified as PTCL–not otherwise specified (PTCL-NOS). PTCL-GATA3, a molecularly defined subtype, associated with T helper 2 (T H 2)–like differentiation and poor prognosis, has frequent co-occurrence of TP53 loss/mutation and heterozygous PTEN loss. CD4+ T cell conditional mouse models with Trp53 mutation/deletion and Pten loss demonstrated mature T cell lymphomas (mTCLs) with T H 2-like transcriptomic and immunophenotypic profiles. Molecular studies revealed that codeletion of Trp53/Pten induced T cell receptor and Janus kinase–signal transducer and activator of transcription signaling, promoting T H 2 differentiation while inhibiting T H 1 differentiation. These findings were validated by CRISPR editing of TP53/PTEN loss in human CD4+ T cells and mechanistically evaluated the p53 binding region in intron-3 of GATA3, resulting in transcriptional repression. Transcriptomic profiles of m-TCLs recapitulated human-PTCL-GATA3 transcriptome and distinguished PTCL-NOS subtypes. Preclinical assessment of m-TCLs with PI3Kγ/δ inhibitors significantly improved survival, supporting a therapeutic approach for the p53-aberrant PTCL-GATA3.

ABSTRACTNodal follicular helper T‐cell (T FH) lymphoma of the angioimmunoblastic (AITL) subtype has a dismal prognosis. Using whole‐exome sequencing (n = 124), transcriptomic (n = 78), and methylation (n = 40) analysis, we identified recurrent mutations in known epigenetic drivers (TET2, DNMT3A, IDH2 R172 ) and novel ones (TET3, KMT2D). TET2, IDH2 R172 , DNMT3A co‐mutated AITLs had poor prognosis (p < 0.0001). Genes regulating T‐cell receptor (TCR) signaling (CD28, PLCG1, VAV1, FYN) or activation (RHOA G17V ) or regulators of the PI3K‐pathway (PIK(3)C members, PTEN, PHLPP1, PHLPP2) were mutated. CD28 mutation/fusion was associated with poor prognosis (p = 0.02). WES of purified, neoplastic T‐cell (CD3+PD1+) demonstrated high concordance with whole tumor biopsies and validated the presence of TET2 and DNMT3A in tumor and non‐lymphoid cells, but other mutations (CD28, RHOA G17V , IDH2 R172 , PLCG1) in neoplastic cells. Integrated DNA‐methylation and mRNA expression analysis revealed epigenetic alterations in genes regulating TCR, cytokines, PI3K‐signaling, and apoptosis. RNA‐seq analysis identified fusion transcripts regulating TCR‐activation (8%), revealed a restricted TCR‐repertoire (α = 87%, β = 72%), and showed the presence of Epstein–Barr virus transcriptome (73%). GEP demonstrated the association of B‐cells or dendritic cells in the tumor milieu with prognosis (p < 0.01). RNA‐seq and WES analysis of 12 AITL‐patient‐derived‐xenografts (PDX) showed that bi‐allelic TET2 and DNMT3A mutations or sub‐clonal mutations (PLCG1, PHLPP2) propagated in sequential passages, and gene signatures related to T FH and T CM (central‐memory) were well‐maintained through passages. Gene expression signatures associated with late PDX passages (3rd–5th) were enriched with proliferation and metabolic reprogramming‐related genes and predicted prognosis in an independent AITL series. Low PHLPP2 mRNA expression predicted poor prognosis (p = 0.05) and engineered PHLPP2 or TET2 loss in CD4+ T‐cells showed enhanced PI(3)K activation, thus uncovering a therapeutic target for clinical trials.

Introduction: Nodal lymphomas of T-follicular helper cells (n-TFHLs) are considered neoplasms of mature TFH cells and are classified by the World Health Organization (WHO) under one umbrella with three subtypes: angioimmunoblastic-type (n-TFHL-AI), nodal TFHL, follicular type (n-TFHL-F), and nodal TFHL, not otherwise specified (n-TFHL-NOS). TET2 mutations in n-TFHL-AI subtype frequently co-occur with IDH2R172 mutations. Methods: To elucidate the cooperative role of TET2 and IDH2R172K mutation in n-TFHL-AI pathogenesis, we generated a murine model with Tet2 loss and IDH2R172K knock-in mutation in CD4+ T cells (Tet2-/-/IDH2R172K) by crossing Tet2Flox/Flox/IDH2R172K mice with Cd4-Cre transgenic mice. This resulted in Tet2 exon 3 deletion and IDH2R172K knock-in mutation specifically in CD4+ T cells. Murine tumors were comprehensively evaluated through immunohistochemistry and flow cytometry. Transcriptomic (RNA-seq), 5-hydroxymethylcytosine (5hmC), and 5-methylcytosine (5mC) profiling, and genomic (whole exome sequencing [WES]) analyses were conducted on both earlier time points (preneoplastic) and neoplastic cells. Functional validation involved proliferation, T-cell polarization, and Western blotting assays. Results: The transcriptomic analysis (p &lt; 0.05) in n-TFHL-AI (double-mutant) cases showed enrichment of TFH-like signature, IL-12-signaling, NF-ΚB, and downregulation of TH1 cell differentiation (including IFNγ signaling). Evaluation of double-mutant mice revealed an earlier tumor onset than Tet2 mutant mice, with a median survival of 1.3 years compared to 1.7 years (p=0.006), respectively. With chronic stimulation by T cell-dependent antigens (sheep red blood cells [SRBCs]), these mice showed B-cell expansion with a 100% incidence of lymphoid neoplasms, in contrast to PBS-treated mice, where myeloid and lymphoid diseases occurred at an equal ratio. Genomic analysis of murine tumors revealed a unique mutation spectrum in metabolic genes (Ata3d3a, Atp1a3, Ppp4r3a, and Nemf), T- and B-cell interaction (Gnai3), Stat pathway (Jak1), and DNA repair (Zfp451). Transcriptomic analysis of tumors showed downregulation of Tbx21, increased TFH-related genes, and increased expression of Bcl6 with downregulation of its known suppressed genes. Transcriptomic analysis of murine CD4+ T cells isolated at 12 weeks revealed downregulation of genes associated with TH1-cell differentiation and Trp53 signaling, concurrent activation of T-cell receptor (TCR) and MAPK signaling, and API-complex. We demonstrated elevated levels of 2-hydroxyglutarate (2-HG) in double-mutant cells, and enasidenib (IDH2R172 inhibitor) treatment significantly decreased 2-HG levels and increased Tbx21 expression. Mutant cells showed a proliferative advantage in IL-2 and IL-21 in vitro conditions, resulting in enhanced TCR, AKT, ERK1/2, p-65, and inactivation of p-FOXO1 protein. While these cells showed a growth advantage in in vitro culturing with TFH conditions, their growth and polarization were impaired in TH1 conditions, indicating a differentiation block. Tet2-/-/IDH2R172K mice (12-week) with SRBCs treatment revealed a significant induction of B and CD4+ T- cell populations compared to wild-type (WT) mice (p=0.01). In vitro co-culturing experiments demonstrated that Tet2-/-/IDH2R172K mutant CD4+ T-cells confer a substantial proliferative advantage to B-cells (p=0.021). Notably, these double-mutant CD4+ T cells exhibited superior proliferation compared to WT CD4+ T-cells when co-cultured with B-cells isolated from WT or double mutant mice (p=0.004). When Tet2-/-/IDH2R172K neoplastic cells with whole tumor were allografted into NOD scid gamma (NSG) mice, lymphoma developed within three months, but also showed numerous B-cells in the TME. This suggests that the propagation of neoplastic T-cells likely require B cell help. Overall, the TFH program remained intact in transplanted cells, and a global decrease in 5hmC levels and an increase in 5mC levels were observed. Conclusion: Tet2-/-/IDH2R172K murine CD4+ T cells exhibited hyperactive TCR signaling, impaired DNA damage response, and NF-ΚB activation as key oncogenic transitions. Both in vitro co-culturing and the allografting of neoplastic Tet2-/-/IDH2R172K cells showed dependence on B-cells, suggesting that interrupting T- and B-cell interaction could be a therapeutic approach.