Waseem Lone

TET2 mutations are frequent in TFH-derived lymphomas, but how epigenetic disruption initiates malignant T cell transformation is unclear. We generated Cd4cre;Tet2FL/FL mice, which developed aggressive T cell lymphoma (m-TCL) with a TFH cell–like immunophenotype. Genome-wide transcriptomics and epigenetic profiling of Tet2−/− CD4+ T cells prior to lymphoma development showed hyperactive TCR, PI3K signaling, and dysregulated TH differentiation program, with proliferation promoting signal transduction at the lymphoma stage. Tet2 loss promoted hyperplasticity under in vitro conditions but favored conditional TFH differentiation. Reduced 5-hmC levels at regulatory genomic elements, resulted in transcriptional rewiring of TFH-associated genes, promoting ICOS(L)-mediated PI3K signaling. TET2-KO human CD4+ T cells showed conserved epigenetic changes with increased proliferation, decreased exhaustion, increased memory marker expression, and clonal expansion with restricted TCR repertoire under in vitro conditions. scRNA-seq revealed a persistent proliferative cluster characterized by elevated stem-like transcriptional features compared with WT counterparts. Tet2−/− m-TCLs allografted into NSG mice showed a significant response to epigenetic (5-azacytidine) and PI3K inhibitors (duvelisib) alone or in combination.

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.

Nodal 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.

High‐grade B‐cell lymphoma not otherwise specified (HGBCL, NOS) has overlapping morphological and genetic features with diffuse large B‐cell lymphoma (DLBCL) and Burkitt lymphoma (BL), leading to uncertainty in its diagnosis and clinical management. Using functional genomic approaches, we previously characterized HGBCL and NOS, that demonstrate gene expression profiling (GEP), and genetic signatures similar to BL. Herein, we characterize distinct HGBCL, NOS, cohort ( n = 55) in adults ( n = 45) and in children ( n = 10), and compared the GEP, genomic DNA copy number (CN), and mutational spectrum with de novo DLBCL ( n = 85) and BL ( n = 52). This subgroup, representing ~60% of HGBCL, NOS, lack gene‐expression signature of BL and double hit/dark zone lymphoma, but express DLBCL like signatures and are characterized by either GCB‐ or ABC‐like mRNA signatures and exhibit higher genomic complexity, similar to de novo DLBCL, and show alteration in genes regulating B‐cell activation ( CD79B , MYD88 , PRDM1 , TBLIXR1 , CARD11 ), epigenome ( KMT2D , TET2 ) and cell cycle transition ( TP53 , ASPM ). However, recurrent mutations in genes often mutated in BL (DDX3X, GNA13, CCND3), but rare in DLBCL, are also present in HGBCL‐NOS, highlighting genetic heterogeneity. Consistent with mutation spectrum, frequent genomic CN alterations in genes regulating B‐cell activation (del‐ PRDM1 , gain‐ BCL6 , ‐ REL , ‐ STAT3 ) and cell cycle regulators (del‐ TP53 , del‐ CDKN2A , del‐ RB1 , gain‐ CCND3 ) were observed. Pediatric cases showed GCB‐DLBCL‐like mRNA signatures, but also featured hallmark mutations of pediatric BL. Frequent oncogenic PIM1 mutations were present in adult HGBCL, NOS. In vitro analyses with pharmacologic or genetic inhibition of PIM1 expression triggered B‐cell activation and NF‐κB‐induced apoptosis, suggesting that PIM1 is a rational therapeutic target.

T-prolymphocytic leukemia (T-PLL) is a rare and mature T-cell malignancy with characteristic chemotherapy-refractory behavior and a poor prognosis. Molecular concepts of disease development have been restricted to protein-coding genes. Recent global microRNA (miR) expression profiles revealed miR-141-3p and miR-200c-3p (miR-141/200c) as two of the highest differentially expressed miRs in T-PLL cells versus healthy donor-derived T cells. Furthermore, miR-141/200c expression separates T-PLL cases into two subgroups with high and low expression, respectively. Evaluating the potential pro-oncogenic function of miR-141/200c deregulation, we discovered accelerated proliferation and reduced stress-induced cell death induction upon stable miR-141/200c overexpression in mature T-cell leukemia/lymphoma lines. We further characterized a miR-141/200c-specific transcriptome involving the altered expression of genes associated with enhanced cell cycle transition, impaired DNA damage responses, and augmented survival signaling pathways. Among those genes, we identified STAT4 as a potential miR-141/200c target. Low STAT4 expression (in the absence of miR-141/200c upregulation) was associated with an immature phenotype of primary T-PLL cells as well as with a shortened overall survival of T-PLL patients. Overall, we demonstrate an aberrant miR-141/200c-STAT4 axis, showing for the first time the potential pathogenetic implications of a miR cluster, as well as of STAT4, in the leukemogenesis of this orphan disease.

PURPOSE: Peripheral T-cell lymphoma (PTCL) includes heterogeneous clinicopathologic entities with numerous diagnostic and treatment challenges. We previously defined robust transcriptomic signatures that distinguish common PTCL entities and identified two novel biologic and prognostic PTCL-not otherwise specified subtypes (PTCL-TBX21 and PTCL-GATA3). We aimed to consolidate a gene expression–based subclassification using formalin-fixed, paraffin-embedded (FFPE) tissues to improve the accuracy and precision in PTCL diagnosis. MATERIALS AND METHODS: We assembled a well-characterized PTCL training cohort (n = 105) with gene expression profiling data to derive a diagnostic signature using fresh-frozen tissue on the HG-U133plus2.0 platform (Affymetrix, Inc, Santa Clara, CA) subsequently validated using matched FFPE tissues in a digital gene expression profiling platform (nCounter, NanoString Technologies, Inc, Seattle, WA). Statistical filtering approaches were applied to refine the transcriptomic signatures and then validated in another PTCL cohort (n = 140) with rigorous pathology review and ancillary assays. RESULTS: In the training cohort, the refined transcriptomic classifier in FFPE tissues showed high sensitivity (> 80%), specificity (> 95%), and accuracy (> 94%) for PTCL subclassification compared with the fresh-frozen–derived diagnostic model and showed high reproducibility between three independent laboratories. In the validation cohort, the transcriptional classifier matched the pathology diagnosis rendered by three expert hematopathologists in 85% (n = 119) of the cases, showed borderline association with the molecular signatures in 6% (n = 8), and disagreed in 8% (n = 11). The classifier improved the pathology diagnosis in two cases, validated by clinical findings. Of the 11 cases with disagreements, four had a molecular classification that may provide an improvement over pathology diagnosis on the basis of overall transcriptomic and morphological features. The molecular subclassification provided a comprehensive molecular characterization of PTCL subtypes, including viral etiologic factors and translocation partners. CONCLUSION: We developed a novel transcriptomic approach for PTCL subclassification that facilitates translation into clinical practice with higher precision and uniformity than conventional pathology diagnosis.

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.

Background: Neoplasms of T-cell or natural killer/T-cell origin account for 10-15% of all non-Hodgkin lymphomas (NHLs) in the United States, and 30% or more of NHLs in African and Asian countries, and tumors from post-thymic or peripheral T-cells are referred to collectively as PTCLs. Recent advances, including approval of brentuximab vedotin (BV), an anti-CD30 monoclonal antibody (mAb) drug conjugate (ADC) with monomethyl auristatin E (MMAE), deacetylase inhibitors (HDACis), and Anaplastic lymphoma kinase (ALK) inhibitors for ALK-positive anaplastic large cell lymphoma (ALCL) have improved outcomes. However, most PTCLs still have a poorer prognosis than comparable B-cell NHLs, and identification of novel targets and drugs retains importance in this area of unmet medical need. Methods: Pre-clinical studies were performed using PTCL and cutaneous T-cell lymphoma (CTCL) cell lines initially in vitro, and then using an in vivo xenograft model. Publically available databases were also leveraged, including the Broad Institute Cancer Cell Line Encyclopedia (CCLE), as well as our own RNA-sequencing (RNA-Seq) data from primary PTCL samples. Results: We examined the cell surface proteome of SUD-HL-1 (ALK+ ALCL), Mac-1 (ALK- ALCL), HH (CTCL), and HuT 78 (Mycosis fungoides with Sézary syndrome) cells by biotinylation and then mass spectrometry, and identified csHSP70 as being consistently expressed in all four lines. Analysis of the CCLE showed that HSP70 mRNA and HSP70 protein was expressed at the highest level in T-cell lymphoma cell lines, and our own RNA-Seq data confirmed HSP70 gene expression was higher in primary PTCL samples, and especially in ALCLs, compared with normal T-cells. To test its promise as a therapeutic target, we generated mAbs to human HSP70 and identified one clone, 239-87, which specifically bound csHSP70 on T-cell NHL cell lines but not on normal peripheral blood-derived mononuclear cells (PBMCs). Next, 239-87 was linked to MMAE to generate an ADC with a drug:antibody ratio of 4, and we confirmed that it was both internalized and then trafficked into acidic vacuoles in SUD-HL-1 cells. The 239-87-MMAE ADC induced a time- and concentration-dependent loss of viability in a panel of PTCL and CTCL cell lines associated with a G2/M arrest and induction of apoptosis, while normal PBMCs were unaffected. Comparisons of the activity of BV with 239-87-MMAE showed that the latter had similar efficacy against SU-DHL-1 and Hut 78 cells in vitro. When cells were propagated under conditions of hypoxia to mimic the tumor microenvironment there was an increase in csHSP70 expression, and the sensitivity of PTCL and CTCL cell lines to the 239-87-MMAE ADC was enhanced. Conversely, an inducible HSP70-targeted short hairpin RNA reduced total and csHSP70 protein expression, and reduced the efficacy of the ADC. Also of note, the HDACi vorinostat enhanced csHSP70 levels, and combinations of vorinostat with the 239-87-MMAE ADC enhanced loss of viability in these cells in a synergistic manner based on combination index analyses. Finally, we prepared an orthotopic in vivo PTCL model by subcutaneously injecting luciferase-labeled Mac-1 cells into C.B-17/IcrHsd-Prkdc ^scid mice. Disease progression occurred rapidly in all mice treated once weekly on days 10, 17, 24, and 31 with an IgG2A isotype mAb, as was the case for 7/8 mice treated with the 239-87-MMAE ADC at 1 mg/kg. In contrast, palpable tumor disappeared in 1/8 mice that received this ADC at 1 mg/kg, and 8/8 and 7/7 mice that received dosing at 5 and 10 mg/kg, respectively (Figure 1A). Tumor recurrence has not been seen at 105 days, including 74 days since the last ADC dose, and the one mouse at 1 mg/kg, and 3 each in the 5 and 10 mg/kg cohorts have had no disease by imaging, while the others have a small residual signal (Figure 1B) that has not progressed for two months. Conclusions: These pre-clinical in vitro and in vivo data support the possibility that csHSP70 could represent a novel therapeutic target for PTCL, and provide a rationale to translate ADCs based on our clone 239-87 mAb to the clinic for patients with advanced ALCL, and potentially other T-cell lymphomas as well.

Purpose: Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non–Hodgkin lymphomas with aggressive clinical behavior. We performed comprehensive miRNA profiling in PTCLs and corresponding normal CD4+ Th1/2 and TFH-like polarized subsets to elucidate the role of miRNAs in T-cell lymphomagenesis. Experimental Design: We used nCounter (NanoString Inc) for miRNA profiling and validated using Taqman qRT-PCR (Applied Biosystems, Inc). Normal CD4+ T cells were polarized into effector Th subsets using signature cytokines, and miRNA significance was revealed using functional experiments. Results: Effector Th subsets showed distinct miRNA expression with corresponding transcription factor expression (e.g., BCL6/miR-19b, -106, -30d, -26b, in IL21-polarized; GATA3/miR-155, miR-337 in Th2-polarized; and TBX21/miR-181a, -331-3p in Th1-polarized cells). Integration of miRNA signatures suggested activation of TCR and PI3K signaling in IL21-polarized cells, ERK signaling in Th1-polarized cells, and AKT–mTOR signaling in Th2-polarized cells, validated at protein level. In neoplastic counterparts, distinctive miRNAs were identified and confirmed in an independent cohort. Integrative miRNA–mRNA analysis identified a decrease in target transcript abundance leading to deregulation of sphingolipid and Wnt signaling and epigenetic dysregulation in angioimmunoblastic T-cell lymphoma (AITL), while ERK, MAPK, and cell cycle were identified in PTCL subsets, and decreased target transcript abundance was validated in an independent cohort. Elevated expression of miRNAs (miR-126-3p, miR-145-5p) in AITL was associated with poor clinical outcome. In silico and experimental validation suggest two targets (miR-126→ SIPR2 and miR-145 → ROCK1) resulting in reduced RhoA-GTPase activity and T–B-cell interaction. Conclusions: Unique miRNAs and deregulated oncogenic pathways are associated with PTCL subtypes. Upregulated miRNA-126-3p and miR-145-5p expression regulate RhoA-GTPase and inhibit T-cell migration, crucial for AITL pathobiology.

One-third of peripheral T-cell lymphomas are “not otherwise specified” (PTCL-NOS), but they have been subdivided into 2 subgroups based on gene expression profiling. Amador and colleagues generated an immunohistochemical algorithm that parallels the molecular separation of PTCL-NOS and provides useful prognostic information.