Notch1-dependent lymphomagenesis is assisted by but does not essentially require pre-TCR signaling

Overexpression of intracellular Notch plays an important role in the generation of human acute lymphoblastic T cell leukemia (T-ALL). In mouse models, it was shown that Notch-dependent T-ALL required pre-TCR signaling. Here we show that pre-TCR signaling is required to condition mice for Notch-dependent transformation but that it is not required to sustain malignant growth of T-ALL. In contrast to previous studies, we found that disease development does not require pre-TCR but that it can be accelerated in Rag2-/- mice by transient mimicking of pre-TCR signals. (Blood. 2006;108:305-310)

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A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma

Blood ◽

10.1182/blood-2019-124641 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1489-1489

Author(s):

Yuichiro Uchida ◽

Makoto Yoshimitsu ◽

Yuhei Kamada ◽

Naosuke Arima ◽

Kenji Ishitsuka

Keyword(s):

T Cell ◽

Signaling Pathway ◽

Research Funding ◽

Jurkat Cells ◽

Cell Phenotype ◽

Acute Type ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Tcr Signaling ◽

Gain Of Function

Introduction: Adult T-cell leukemia/lymphoma (ATL) is an intractable T cell malignancy with regulatory T cell phenotype, which caused by long-term infection of human T-cell leukemia virus type-1 (HTLV-1). In addition to HTLV-1-derived oncogenic proteins such as Tax and HBZ, genomic aberrations including gain-of-function alterations in genes related to T-cell receptor (TCR) signaling pathway have been implicated in the pathogenesis of ATL. RLTPR is essential for CD28 co-stimulation in human T cells and loss-of-function mutations in RLTPR is reported to reduce proportions of regulatory T cells. The RLTPR p.Q575E has been reported as a recurrent mutation in cutaneous T cell lymphoma1. Here, we show that RLTPR p.Q575E is a novel recurrent gain-of-function mutation related to T-cell receptor co-stimulatory pathway in patients with acute type ATL. Methods To elucidate the genomic basis of ATL, we performed whole exome sequence (WES) in peripheral blood mononuclear cells (CD4/CD25 positive cells were sorted if ATL cell were not abundant) from 47 patients with acute-type ATL. Sequences were aligned against the reference genome (GRCh37/hg19) by using TMAP Alignment. Sanger sequencing was performed to confirm the variant obtained after WES genotyping for some genes including RLTPR. Clinical information regarding ATL cell phenotype, treatment and clinical course were also collected. To analyze the function of RLTPR Q575E, the human RLTPR isoform 3 wild type (WT) (most abundant isotype in lymphoid cells, RLTPR-WT hereafter) and RLTPR isoform 3 Q575E (RLTPR Q575E hereafter) expression retroviral vector were constructed. RLTPR isoform 1 WT and RLTPR isoform 1 Q575E were used as control. RLPTR WT or RLTPR Q575E transduced Jurkat cells were generated by retroviral transduction. NFAT, NF- κB or AP-1 promoter activity was measured as luciferase activity. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin calcium salt for TCR stimulation and CD86 Fc chimera for CD28 stimulation. IL-2 mRNA was quantitated by using Taqman Gene Expression Assays. Results Exome analysis from 47 acute-type ATL samples revealed gene alterations precipitated in TCR signaling pathway (CARD11;30.0%, PLCG1;27.7%, PRKCB;21.3%, STAT3;21.3%, VAV1;19.1%, NOTCH1;19.1%, RELA;12.8%, CNSK1A1;8.5%, IRF4;6.4%, FYN;2.1%, CBLB;2.1%, IKBKB;2.1%). In addition to these previously reported driver genes, a novel mutation, RLTPR Q575E was discovered in 4 out of 47 acute-type ATL samples (8.5%) with median variant allele frequency 0.52 (range 0.11-0.68). Although RLTPR Q575E has been reported in cutaneous T cell lymphoma, 3 out of 4 ATL patients carrying RLTPR Q575E mutation have no skin involvement by ATL. ATL patients carrying RLTPR Q575E were also harboring CARD11 (75%), PLCG1 (25%), PRKCB (25%), or IKBKB (25%) mutations, which are related to TCR/NF-κB signaling pathway. We next performed luciferase reporter assay to evaluate NF-κB activity in transfected 293T cells with RLTPR Q575E cDNA to explore its function. 293T cells transfected with RLTPR Q575E cDNA has higher NF-κB activity than those transfected with RLTPR wild type cDNA. No significant increased promoter activity of AP-1 or NFAT was observed with RLTPR Q575E. IL-2 mRNA was significantly increased in RLTPR Q575E transduced Jurkat cells under PMA/ionomycin and CD86 co-stimulation. We immunoprecipitated FLAG-tagged RLTPR WT or RLTPR Q575E and blotted for CARD11. We found that the RLTPR Q575E increases interaction of RLTPR with CARD11. Previously identified gene alterations such as CARD11 and PRKCB in ATL have been reported as Tax interactome, thus we further analyzed the interaction of RLTPR with Tax. We immunoprecipitated FLAG-tagged WT RLTPR or RLTPR Q575E and blotted for Tax. We found that the RLTPR WT and Q575E increases interaction of RLTPR with Tax. Conclusions In patients with acute-type ATL, we have identified RLTPR Q575E, a novel gain of function mutation, and functionally validated this mutation. This mutation has minimal effect without TCR co-stimulation, but patients with this mutation harboring gain-of-function mutations in TCR signaling molecules. We also found direct interaction between Tax and RLTPR. In the presence of TCR activation by Tax or gain-of-function mutations in this signaling pathway, RLTPR Q575E mutation selectively upregulates the NF-κB signaling pathway and confers the oncogenic effect on the pathogenesis of ATL. Reference 1 Joonhee Park, Jingyi Yang, Alexander T. Wenzel, Akshaya Ramachandran, Wung J. Lee, Jay C. Daniels, Juhyun Kim, Estela Martinez-Escala, Nduka Amankulor, Barbara Pro, Joan Guitart, Marc L. Mendillo, Jeffrey N. Savas, Titus J. Boggon, Jaehyuk Choi; Genomic analysis of 220 CTCLs identifies a novel recurrent gain-of-function alteration in RLTPR (p.Q575E). Blood 2017; 130 (12): 1430-1440. doi: https://doi.org/10.1182/blood-2017-02-768234 Disclosures Yoshimitsu: Novartis: Speakers Bureau; Bristol-Myer-Squibb,: Speakers Bureau; Shire: Speakers Bureau; Takeda: Speakers Bureau; Chugai: Speakers Bureau; Sanofi: Speakers Bureau. Ishitsuka:Novartis: Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; Otsuka Pharmaceutical: Honoraria; Janssen Pharmaceutical: Honoraria; Taiho Pharmaceutical: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Shire: Honoraria; mundiharma: Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Teijin Pharma: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria; Teijin Pharma: Research Funding; Eli Lilly: Research Funding; Eisai: Honoraria, Research Funding; Mochida: Honoraria, Research Funding; sanofi: Honoraria; Pfizer: Honoraria; Alexion: Honoraria; Genzyme: Honoraria; Astellas Pharma: Honoraria, Research Funding; Asahi kasei: Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; Yakult: Research Funding; MSD: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; MSD: Research Funding; Asahi kasei: Research Funding; Yakult: Research Funding; Bristol-Myers Squibb: Honoraria; Shire: Honoraria; Eli Lilly: Research Funding.

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Frequent Activating Somatic Alterations in T-Cell Receptor / NF-κb Signaling in Adult T-Cell Leukemia/Lymphoma

Blood ◽

10.1182/blood.v126.23.113.113 ◽

2015 ◽

Vol 126 (23) ◽

pp. 113-113 ◽

Cited By ~ 1

Author(s):

Keisuke Kataoka ◽

Yasunobu Nagata ◽

Akira Kitanaka ◽

Yuichi Shiraishi ◽

Jun-ichirou Yasunaga ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Research Funding ◽

Cell Receptor ◽

T Cell Leukemia ◽

Rna Seq ◽

Cell Leukemia ◽

Tcr Signaling ◽

Adult T Cell Leukemia ◽

Somatic Alterations

Abstract Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm of largely unknown genetic basis, which is associated with human T-cell leukemia virus type-1 (HTLV-1) infection. To delineate a genetic landscape of somatic alterations in ATL, we have performed an integrated genetic study, in which whole-genome/exome (WGS/WES) and transcriptome sequencing (RNA-seq) was performed for a cohort of 83 paired ATL samples, followed by extensive validation using targeted sequencing of detected mutations in 370 follow-up samples. A striking feature of driver lesions in ATL was their strong enrichment in the components of T-cell receptor (TCR) / NF-κB pathway. Accounting for more than 90% of ATL cases, these lesions were characterized by the predominance of activating alterations, including hotspot missense mutations in PLCG1 (36%), PRKCB (33%), CARD11 (24%), VAV1 (18%), IRF4 (14%) and FYN (4%). Among these, most frequently mutated was PLCG1, which encodes phospholipase C γ1 (PLCγ1), a key regulator of the proximal TCR signaling. Besides the S345F and S520F mutations recently reported in cutaneous T-cell lymphoma, we identified an additional hotspot mutations (R48W, E1163K, and D1165H). The second most frequently mutated gene was PRKCB, encoding a member of the protein kinase C (PKC) family of proteins (PKCβ), a pivotal signaling molecule downstream of PLCγ. The frequent mutations of PKCβ were unexpected, because it is PKCθ that has been implicated in TCR signaling, whereas PKCβ has been more focused in the context of B-cell receptor signaling. Approximately 93% of the PRKCB mutations were confined to the catalytic domain with a prominent hotspot at D427, suggesting gain-of-function nature of these mutations. Consistent with this, when transduced with the D427N PKCβ mutant, HEK293T and/or Jurkat cells showed increased membrane translocation after PMA/Ionomycin-stimulation, enhanced IKK phosphorylation and p65 nuclear translocation, and augmented NF-κB transcription, compared to wild-type PKCβ-transduced cells. Thus, these PRKCB mutations are the first activating mutations of this family identified in human cancers. Downstream to PKC lies CARD11, a scaffolding protein required for antigen receptor-induced NF-κB activation. Although previously reported in B-cell lymphomas, CARD11 mutations were more common in ATL (24%). In B-cell lymphomas, mutations are largely limited to the coiled-coil (CC) domain, whereas in ATL, they were clustered not only within the CC domain, but also within the PKC-responsive inhibitory domain, showing a prominent mutational hotspot at E626. The inhibitory domain has been implicated in autoinhibition, whose deletion leads to constitutive activation of CARD11. Intriguingly, WGS identified small intragenic deletions confined to this domain (exons 14-17) in 4 cases (8%) without canonical mutations, and RNA-seq confirmed the skipping of the corresponding exons in these cases. Remarkably, CARD11 mutation significantly co-occurred with PRKCBmutations, suggesting potential functional synergism between these lesions. Actually, overexpression of wild-type CARD11 induced NF-κB activation, which was further augmented by E626K mutation. Similarly, when both CARD11 (E626K) and PRKCB (D427N) mutants were co-expressed, more enhanced NF-κB activation was observed. RNA-seq and follow-up RT-PCR screening also identified novel gene fusions in TCR / NF-κB pathway: five CTLA4-CD28 and three ICOS-CD28 fusions were observed in seven (7%) of the 105 cases examined, of whom one patient carried both chimeric fusions. WGS revealed tandem duplications of 2q33.2 segments containing CD28, CTLA4, and ICOS, compatible with the corresponding fusion transcripts. B7/CD28 co-signaling molecules, including CD28, CTLA4, and ICOS co-receptors, play pivotal roles in positive and negative regulations of TCR signaling. All the predicted chimeric proteins had the cytoplasmic part of CD28, and are expected to be expressed under the control of the regulatory element of CTLA4 or ICOS, likely leading to prolonged expression of CD28 co-stimulator. Our findings suggest that deregulated TCR / NF-κB pathway caused by genetic alterations is a hallmark of ATL pathogenesis. The predominance of gain-of-function mutations in this pathway offers good opportunities for exploiting these mutations for the targets of novel drugs to better manage patients. Disclosures Tobinai: Gilead Sciences: Research Funding. Miyazaki:Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

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Advanced Pathogenetic Concepts in T-Cell Prolymphocytic Leukemia and Their Translational Impact

Frontiers in Oncology ◽

10.3389/fonc.2021.775363 ◽

2021 ◽

Vol 11 ◽

Author(s):

Till Braun ◽

Annika Dechow ◽

Gregor Friedrich ◽

Michael Seifert ◽

Johanna Stachelscheid ◽

...

Keyword(s):

T Cell ◽

Transcriptional Activation ◽

Current Knowledge ◽

Disease Model ◽

Cell Receptor ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Prolymphocytic Leukemia ◽

Tcr Signals ◽

Family Based

T-cell prolymphocytic leukemia (T-PLL) is the most common mature T-cell leukemia. It is a typically aggressively growing and chemotherapy-resistant malignancy with a poor prognosis. T-PLL cells resemble activated, post-thymic T-lymphocytes with memory-type effector functions. Constitutive transcriptional activation of genes of the T-cell leukemia 1 (TCL1) family based on genomic inversions/translocations is recognized as a key event in T-PLL’s pathogenesis. TCL1’s multiple effector pathways include the enhancement of T-cell receptor (TCR) signals. New molecular dependencies around responses to DNA damage, including repair and apoptosis regulation, as well as alterations of cytokine and non-TCR activation signaling were identified as perturbed hallmark pathways within the past years. We currently witness these vulnerabilities to be interrogated in first pre-clinical concepts and initial clinical testing in relapsed/refractory T-PLL patients. We summarize here the current knowledge on the molecular understanding of T-PLL’s pathobiology and critically assess the true translational progress around this to help appraisal by caregivers and patients. Overall, the contemporary concepts on T-PLL’s pathobiology are condensed in a comprehensive mechanistic disease model and promising interventional strategies derived from it are highlighted.

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Pre-TCR expression cooperates with TEL-JAK2 to transform immature thymocytes and induce T-cell leukemia

Blood ◽

10.1182/blood-2006-09-048801 ◽

2006 ◽

Vol 109 (9) ◽

pp. 3972-3981 ◽

Cited By ~ 22

Author(s):

Nuno R. dos Santos ◽

David S. Rickman ◽

Aurélien de Reynies ◽

Françoise Cormier ◽

Maryvonne Williame ◽

...

Keyword(s):

T Cell ◽

Present Report ◽

Gene Profiling ◽

Leukemic Cells ◽

Specific Cell ◽

Human Leukemia ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Tcr Signaling ◽

Immature Thymocytes

Abstract The TEL-JAK2 gene fusion, which has been identified in human leukemia, encodes a chimeric protein endowed with constitutive tyrosine kinase activity. TEL-JAK2 transgenic expression in the mouse lymphoid lineage results in fatal and rapid T-cell leukemia/lymphoma. In the present report we show that T-cell leukemic cells from EμSRα-TEL-JAK2 transgenic mice present an aberrant CD8+ differentiation phenotype, as determined by the expression of stage-specific cell surface markers and lineage-specific genes. TEL-JAK2 transforms immature CD4−CD8− double-negative thymocytes, as demonstrated by the development of T-cell leukemia with full penetrance in a Rag2-deficient genetic background. This disease is similar to the bona fide TEL-JAK2 disease as assessed by phenotypic and gene profiling analyses. Pre-TCR signaling synergizes with TEL-JAK2 to transform immature thymocytes and initiate leukemogenesis as shown by (1) the delayed leukemia onset in Rag2-, CD3ϵ- and pTα-deficient mice, (2) the occurrence of recurrent chromosomal alterations in pre-TCR–deficient leukemia, and (3) the correction of delayed leukemia onset in Rag2-deficient TEL-JAK2 mice by an H-Y TCRαβ transgene that mimics pre-TCR signaling. Although not affecting leukemia incidence and mouse survival, TCRαβ expression was shown to facilitate leukemic cell expansion in secondary lymphoid organs.

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