Jochum, Dylan T.

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.

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 < 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.

Introduction Peripheral T-cell lymphoma (PTCL) encompasses a heterogeneous group of clinically aggressive entities. ~30% of cases cannot be further classified and are designated as PTCL - not otherwise specified (NOS). We delineated two novel molecular subgroups among PTCL-NOS with distinct clinical and biological features: PTCL-GATA3 which is characterized by high expression of GATA3, a master regulator of T-helper-2-cell (TH2) differentiation, and its target genes (i.e., CCR4, IL4, IL13) and PTCL-TBX21 which is characterized by high expression of TBX21, a master regulator of TH1 differentiation, and its target genes (i.e., CXCR3, IFNG), with the GATA3 subtype showing significantly worse clinical outcomes (overall survival). Nonsense mutations in CCR4 are found predominately in PTCL-GATA3, often within the cytoplasmic domain and gain-of-function in nature. Methods Mutation and copy number data were generated from whole exome sequencing (TFH phenotype excluded following LLMPP pathology review; n = 153). Single nucleotide variants were determined using Mutect2 and Varsvan2 using in-house filtering. Copy number (CN) profiles were generated using CNVkit. Four CCR4 cytoplasmic domain mutants and controls (cytoplasmic domain knockout [CDKO], wild-type CCR4 overexpression (CCR4-WT), and empty vector [EV]) were transduced into healthy-donor CD4+ T cells (n = 2). Polarization studies were generated using TH1- or TH2-associated cytokine exposure. Surface plasmon resonance was used to determine the binding affinity of CCR4 proteins to CCL17 and CCL22. In silico docking studies were done through Schrödinger's BioLuminate. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay was performed through co-incubation of NK92 (CD16 and CD56) and CCR4 edited primary CD4+ T-cells (stained with calcein AM) for 2 hours at a 5:1 NK:T cell ratio by 7-AAD flow cytometry. Results PTCL-GATA3 had a higher frequency of CN aberrations (e.g. TP53, PTEN CN loss) and a more aberrant genome (p < 0.001) than PTCL-TBX21, including mutations with genes associated with DNA repair and damage (TP53, ATM) and T-cell differentiation (CCR4). CCR4 mutations were virtually mutually exclusive with TP53 mutations, and these cases showed higher CCR4 mRNA and protein expression than CCR4-WT cases (p < 0.05). Primary CD4+ T-cells with ectopic expression of the CCR4 variants, especially CCR4-Q330X, did not require CD3/CD28 for proliferation, bypassing CD3/CD28 ligation for T-cell activation. In addition, proliferation rates were similar between TH2-conditional media and normal media, but proliferation and TBX21 expression in TH1-conditional media were inhibited, implying a novel role of CCR4 in T-cell receptor (TCR) activation and TH-differentiation. CCR4 variant proteins showed higher binding affinity for their natural ligands, CCL22 and CCL17, with mutants exhibiting tighter binding to both ligands (1.5- to 3-fold decrease in KD) by surface plasmon resonance and in silico modeling studies. The in silico models demonstrated mutant CCR4 bound mogamulizumab tighter than CCR4-WT, consistent with a higher ADCC-induced apoptosis rate, highest with CCR4-Q330X mutation. CCR4-Y331X exhibited a worse docking score compared to CCR4-WT and a lower ADCC-induced apoptosis rate. Conclusions Our findings reveal distinct genetic characterizations including aberrant TP53 and PI3K signaling and CCR4 mutations in PTCL-GATA3. These CCR4 mutations are shown to have a key role in PTCL-GATA3 pathogenesis by dysregulating TH-differentiation and aberrantly activating the TCR. CCR4 mutation promotes the TH2 phenotype by blocking TH1 differentiation. CCR4 mutant proteins demonstrated an increased binding affinity towards its known ligands, CCL17 and CCL22, and mogamulizumab, consistent with previous studies exhibiting mogamulizumab efficacy against CCR4 mutations. However, the most frequent CCR4 mutant, Y331X, showed worse binding compared to other CCR4 mutants or CCR4-WT, a mutation that showed Mogamulizumab-resistance in a prior CTCL study as well as in an in vitro ADCC assay. The results emphasize aberrant pathways implicated by genomic and clinical evaluation of two novel PTCL subtypes, as well as support the novel role of mutant CCR4 in enhancing T-cell lymphomagenesis and T-helper differentiation, highlighting multiple therapeutic targets.

Abstract Peripheral T-cell lymphomas (PTCLs) are heterogenous T-cell neoplasms often associated with epigenetic dysregulation. We investigated de novo DNA methyltransferase 3A (DNMT3A) mutations in common PTCL entities, including angioimmunoblastic T-cell lymphoma and novel molecular subtypes identified within PTCL–not otherwise specified (PTCL-NOS) designated as PTCL-GATA3 and PTCL-TBX21. DNMT3A-mutated PTCL-TBX21 cases showed inferior overall survival (OS), with DNMT3A-mutated residues skewed toward the methyltransferase domain and dimerization motif (S881–R887). Transcriptional profiling demonstrated significant enrichment of activated CD8+ T-cell cytotoxic gene signatures in the DNMT3A-mutant PTCL-TBX21 cases, which was further validated using immunohistochemistry. Genomewide methylation analysis of DNMT3A-mutant vs wild-type (WT) PTCL-TBX21 cases demonstrated hypomethylation in target genes regulating interferon-γ (IFN-γ), T-cell receptor signaling, and EOMES (eomesodermin), a master transcriptional regulator of cytotoxic effector cells. Similar findings were observed in a murine model of PTCL with Dnmt3a loss (in vivo) and further validated in vitro by ectopic expression of DNMT3A mutants (DNMT3A-R882, -Q886, and -V716, vs WT) in CD8+ T-cell line, resulting in T-cell activation and EOMES upregulation. Furthermore, stable, ectopic expression of the DNMT3A mutants in primary CD3+ T-cell cultures resulted in the preferential outgrowth of CD8+ T cells with DNMT3AR882H mutation. Single-cell RNA sequencing(RNA-seq) analysis of CD3+ T cells revealed differential CD8+ T-cell subset polarization, mirroring findings in DNMT3A-mutated PTCL-TBX21 and validating the cytotoxic and T-cell memory transcriptional programs associated with the DNMT3AR882H mutation. Our findings indicate that DNMT3A mutations define a cytotoxic subset in PTCL-TBX21 with prognostic significance and thus may further refine pathological heterogeneity in PTCL-NOS and suggest alternative treatment strategies for this subset.