T-cell lymphoma patient harboring BCL11B mutations had favorable overall survival.

BACKGROUND: Molecular genetics serve a critical role in constructing risk stratification for hematological malignancies, but T-cell lymphoma (TCL) still lacks molecular genetic information for supplement risk stratification in predicting the prognosis of TCL patients. In the present study, we characterized the mutation patterns of B-cell leukemia/lymphoma 11B gene (BCL11B) and its prognostic importance in TCL patients. METHODS: BCL11B mutations were characterized based on the data from two datasets, one is from our clinical center (GDPH dataset, n = 79) and the other is from COSMIC dataset (n = 154). RESULTS: The overall mutation rate of BCL11B was 6.4% (15/233) in TCL, and there were no hotspot mutation sites in TCL. Among these mutations, the missense and splice site mutation were significantly prominent. Moreover, TCL patients harboring BCL11B mutations had a favorable overall survival (OS) in our center (GDPH dataset) (adjusted hazard ratio [HR] = .001, p = 0.109), although there were not yet significantly statistical at this point. In addition, TCL patients harboring BCL11B mutation had a longer 5-year restricted mean survival time (RMST) than those without a BCL11B mutation (60 vs. 32 months). Notably, BCL11B mutations were not associated with TCL entities having better prognosis. CONCLUSIONS: BCL11B mutations were associated with favorable clinical outcome for TCL patients; it might be considered as a novel biomarker for TCL prognostic stratification.

TMEM244 Is a Long Non-Coding RNA Necessary for CTCL Cell Growth.

Transmembrane protein 244 (TMEM244) was annotated to be a member of the TMEM family, which are is a component of cell membranes and is involved in many cellular processes. To date, the expression of the TMEM244 protein has not been experimentally confirmed, and its function has not been clarified. Recently, the expression of the TMEM244 gene was acknowledged to be a diagnostic marker for Sézary syndrome, a rare cutaneous T-cell lymphoma (CTCL). In this study, we aimed to determine the role of the TMEM244 gene in CTCL cells. Two CTCL cell lines were transfected with shRNAs targeting the TMEM244 transcript. The phenotypic effect of TMEM244 knockdown was validated using green fluorescent protein (GFP) growth competition assays and AnnexinV/7AAD staining. Western blot analysis was performed to identify the TMEM244 protein. Our results indicate that TMEM244 is not a protein-coding gene but a long non-coding RNA (lncRNA) that is necessary for the growth of CTCL cells.

High expression of TMEM244 is associated with poor overall survival of patients with T-cell lymphoma.

T-cell lymphoma (TCL) is an aggressive and genetically heterogeneous malignancy with adverse clinical outcomes; thus, it is worth exploring biomarkers for risk stratification. Previous studies have demonstrated that transmembrane protein 244 gene (TMEM244) is ectopically expressed in Sézary syndrome (SS). In this study, the expression level of TMEM244 and its prognostic value for TCL patients was explored by analyzing RNA-seq data of two large datasets (GSE132550 and GSE113113) containing 129 TCL patients and 13 healthy individuals (HIs) from the Gene Expression Omnibus (GEO) database, the PRJCA002270 dataset containing 124 patients with T-cell acute lymphoblastic leukemia (T-ALL) from the BioProject database, and peripheral blood (PB) samples of 24 TCL and 29 T-ALL patients, as well as 11 normal CD3 + T-cells from our clinical center (JNU). The results suggested that TMEM244 was significantly up-regulated in TCL patients compared with normal CD3 + T-cells or T-ALL in the JNU, GSE132550 and GSE113113 datasets (P < 0.05). However, TMEM244 shows no expression in patients with T-ALL in the JNU-T-ALL and PRJCA002270 datasets. The receiver operating characteristic (ROC) curve analysis indicated that TMEM244 expression had a very high accuracy in diagnosing TCL compared with T-ALL (area under the curve (AUC): 99.4%; P < 0.001). Importantly, high TMEM244 expression was significantly associated with poor OS and shorter 5-year restricted mean survival time (RMST) in TCL patients, especially those treated with chemotherapy. In summary, TMEM244 is also expressed in other types of TCL besides SS, but not in T-ALL. High TMEM244 expression is associated with poor OS in TCL patients, which might be a novel biomarker for prognostic stratification in TCL patients and facilitate the design of novel therapies.

Generation of Inducible BCL11B Knockout in TAL1/LMO1 Transgenic Mouse T Cell Leukemia/Lymphoma Model.

The B-cell CLL/lymphoma 11B gene (BCL11B) plays a crucial role in T-cell development, but its role in T-cell malignancies is still unclear. To study its role in the development of T-cell neoplasms, we generated an inducible BCL11B knockout in a murine T cell leukemia/lymphoma model. Mice, bearing human oncogenes TAL BHLH Transcription Factor 1 (TAL1; SCL) or LIM Domain Only 1 (LMO1), responsible for T-cell acute lymphoblastic leukemia (T-ALL) development, were crossed with BCL11B floxed and with CRE-ER/lox mice. The mice with a single oncogene BCL11Bflox/floxCREtg/tgTAL1tg or BCL11Bflox/floxCREtg/tgLMO1tg were healthy, bred normally, and were used to maintain the mice in culture. When crossed with each other, >90% of the double transgenic mice BCL11Bflox/floxCREtg/tgTAL1tgLMO1tg, within 3 to 6 months after birth, spontaneously developed T-cell leukemia/lymphoma. Upon administration of synthetic estrogen (tamoxifen), which binds to the estrogen receptor and activates the Cre recombinase, the BCL11B gene was knocked out by excision of its fourth exon from the genome. The mouse model of inducible BCL11B knockout we generated can be used to study the role of this gene in cancer development and the potential therapeutic effect of BCL11B inhibition in T-cell leukemia and lymphoma.

The Usefulness of Cell-Based and Liquid-Based Urine Tests in Clarifying the Diagnosis and Monitoring the Course of Urothelial Carcinoma. Identification of Novel, Potentially Actionable, RB1 and ERBB2 Somatic Mutations.

Bladder cancer is one of the most common cancers in global statistics. One of the issues associated with this disease is the high incidence of cases with delayed diagnosis and what factors correlate with worse treatment outcomes. A possible reason for this may be the rather limited availability of non-invasive diagnostic tools. This short communication presents a case of a 68 year old male patient after an ineffective therapy, carried on for several years with symptoms commonly associated with prostate overgrowth that masked a carcinoma in situ of the urinary bladder. Implementation of several diagnostic techniques, including urine sediment cytology, immunocytochemistry, the fluorescence in situ hybridisation technique, the Bladder EpiCheck test and whole-genome sequencing, enabled the establishment of a correct diagnosis, implementation of appropriate treatment and provision of patient-friendly monitoring. The described case emphasises the usefulness of cell-based and liquid-based urine tests in bladder cancer diagnostic procedures.

Inhibition of BCL11B induces downregulation of PTK7 and results in growth retardation and apoptosis in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of leukemia with poor prognosis, and biomarkers and novel therapeutic targets are urgently needed for this disease. Our previous studies have found that inhibition of the B-cell leukemia/lymphoma 11B (BCL11B) gene could significantly promote the apoptosis and growth retardation of T-ALL cells, but the molecular mechanism underlying this effect remains unclear. This study intends to investigate genes downstream of BCL11B and further explore its function in T-ALL cells. We found that PTK7 was a potential downstream target of BCL11B in T-ALL. Compared with the healthy individuals (HIs), PTK7 was overexpressed in T-ALL cells, and BCL11B expression was positively correlated with PTK7 expression. Importantly, BCL11B knockdown reduced PTK7 expression in T-ALL cells. Similar to the effects of BCL11B silencing, downregulation of PTK7 inhibited cell proliferation and induced apoptosis in Molt-4 cells via up-regulating the expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and p27. Altogether, our studies suggest that PTK7 is a potential downstream target of BCL11B, and downregulation of PTK7 expression via inhibition of the BCL11B pathway induces growth retardation and apoptosis in T-ALL cells.

Hypomethylation of the promoter region drives ectopic expression of TMEM244 in Sézary cells.

Sézary syndrome (SS) is an aggressive form of cutaneous T-cell lymphoma (CTCL) characterized by the presence of circulating malignant CD4+ T cells (Sézary cells) with many complex changes in the genome, transcriptome and epigenome. Epigenetic dysregulation seems to have an important role in the development and progression of SS as it was shown that SS cells are characterized by widespread changes in DNA methylation. In this study, we show that the transmembrane protein coding gene TMEM244 is ectopically expressed in all SS patients and SS-derived cell lines and, to a lower extent, in mycosis fungoides and in a fraction of T-cell lymphomas, but not in B-cell malignancies and mononuclear cells of healthy individuals. We show that in patient samples and in the T-cell lines TMEM244 expression is negatively correlated with the methylation level of its promoter. Furthermore, we demonstrate that TMEM244 expression can be activated in vitro by the CRISPR-dCas9-induced specific demethylation of TMEM244 promoter region. Since both, TMEM244 expression and its promoter demethylation, are not detected in normal lymphoid cells, they can be potentially used as markers in Sézary syndrome and some other T-cell lymphomas.

Novel targeted therapies of T cell lymphomas.

T cell lymphomas (TCL) comprise a heterogeneous group of non-Hodgkin lymphomas (NHL) that often present at an advanced stage at the time of diagnosis and that most commonly have an aggressive clinical course. Treatment in the front-line setting is most often cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP-like regimens, which are effective in B cell lymphomas, but in TCL are associated with a high failure rate and frequent relapses. Furthermore, in contrast to B cell NHL, in which substantial clinical progress has been made with the introduction of monoclonal antibodies, no comparable advances have been seen in TCL. To change this situation and improve the prognosis in TCL, new gene-targeted therapies must be developed. This is now possible due to enormous progress that has been made in the last years in the understanding of the biology and molecular pathogenesis of TCL, which enables the implementation of the research findings in clinical practice. In this review, we present new therapies and current clinical and preclinical trials on targeted treatments for TCL using histone deacetylase inhibitors (HDACi), antibodies, chimeric antigen receptor T cells (CARTs), phosphatidylinositol 3-kinase inhibitors (PI3Ki), anaplastic lymphoma kinase inhibitors (ALKi), and antibiotics, used alone or in combinations. The recent clinical success of ALKi and conjugated anti-CD30 antibody (brentuximab-vedotin) suggests that novel therapies for TCL can significantly improve outcomes when properly targeted.

Mouse models to study genes involved in hematological malignancies.

Hematological malignancies, including leukemia and lymphoma, consist a group of highly heterogeneous neoplasms characterized by numerous genetic lesions specific for the type of the disease. In order to understand, the role of a particular alteration in the development of a malignancy functional studies have to be carried out in vitro, in cell lines derived from primary cancer cells. Further efforts to understand the mechanisms underlying blood disorders including malignant transformation and progression relies on model organism research. Numerous transgenic mouse models, carrying human oncogenes have been generated resembling distinct types of hematological disorders. Recent technological advances revolutionized the generation of animal models making it much easier, faster, and precise. The introduction of the CRISPR-Cas9 technology allows for rapid generation of novel knockout or transgenic animals, and the development of conditional site- and time-specific Cre-Lox gene targeting technology, allows studying the function of genes which are relevant to normal hematopoiesis and development of hematological malignancies, but lethal when knocked out in embryonic cells. Besides the studies on gene function, mouse models of human leukemia allow for discovery and testing of novel antileukemic drugs. These new technologies are deepening our understanding of disease pathophysiology and treatment resistance, as well as are leading to novel therapeutic strategies for improved outcomes in patients.

Genome-Wide Analysis of Interchromosomal Interaction Probabilities Reveals Chained Translocations and Overrepresentation of Translocation Breakpoints in Genes in a Cutaneous T-Cell Lymphoma Cell Line.

In classical models of tumorigenesis, the accumulation of tumor promoting chromosomal aberrations is described as a gradual process. Next-generation sequencing-based methods have recently revealed complex patterns of chromosomal aberrations, which are beyond explanation by these classical models of karyotypic evolution of tumor genomes. Thus, the term chromothripsis has been introduced to describe a phenomenon, where temporarily and spatially confined genomic instability results in dramatic chromosomal rearrangements limited to segments of one or a few chromosomes. Simultaneously arising and misrepaired DNA double-strand breaks are also the cause of another phenomenon called chromoplexy, which is characterized by the presence of chained translocations and interlinking deletion bridges involving several chromosomes. In this study, we demonstrate the genome-wide identification of chromosomal translocations based on the analysis of translocation-associated changes in spatial proximities of chromosome territories on the example of the cutaneous T-cell lymphoma cell line Se-Ax. We have used alterations of intra- and interchromosomal interaction probabilities as detected by genome-wide chromosome conformation capture (Hi-C) to infer the presence of translocations and to fine-map their breakpoints. The outcome of this analysis was subsequently compared to datasets on DNA copy number alterations and gene expression. The presence of chained translocations within the Se-Ax genome, partly connected by intervening deletion bridges, indicates a role of chromoplexy in the etiology of this cutaneous T-cell lymphoma. Notably, translocation breakpoints were significantly overrepresented in genes, which highlight gene-associated biological processes like transcription or other gene characteristics as a possible cause of the observed complex rearrangements. Given the relevance of chromosomal aberrations for basic and translational research, genome-wide high-resolution analysis of structural chromosomal aberrations will gain increasing importance.

The N-Terminal CCHC Zinc Finger Motif Mediates Homodimerization of Transcription Factor BCL11B.

The BCL11B gene encodes a Krüppel-like, sequence-specific zinc finger (ZF) transcription factor that acts as either a repressor or an activator, depending on its posttranslational modifications. The importance of BCL11B in numerous biological processes in multiple organs has been well established in mouse knockout models. The phenotype of the first de novo monoallelic germ line missense mutation in the BCL11B gene (encoding N441K) strongly implies that the mutant protein acts in a dominant-negative manner by neutralizing the unaffected protein through the formation of a nonfunctional dimer. Using a Förster resonance energy transfer-assisted fluorescence-activated cell sorting (FACS-FRET) assay and affinity purification followed by mass spectrometry (AP-MS), we show that the N-terminal CCHC zinc finger motif is necessary and sufficient for the formation of the BCL11B dimer. Mutation of the CCHC ZF in BCL11B abolishes its transcription-regulatory activity. In addition, unlike wild-type BCL11B, this mutant is incapable of inducing cell cycle arrest and protecting against DNA damage-driven apoptosis. Our results confirm the BCL11B dimerization hypothesis and prove its importance for BCL11B function. By mapping the relevant regions to the CCHC domain, we describe a previously unidentified mechanism of transcription factor homodimerization.

Genetic rearrangements result in altered gene expression and novel fusion transcripts in Sézary syndrome.

Sézary syndrome (SS) is an aggressive, leukemic cutaneous T-cell lymphoma variant. Molecular pathogenesis of SS is still unclear despite many studies on genetic alterations, gene expression and epigenetic regulations. Through whole genome and transcriptome next generation sequencing nine Sézary syndrome patients were analyzed in terms of copy number variations and rearrangements affecting gene expression. Recurrent copy number variations were detected within 8q (MYC, TOX), 17p (TP53, NCOR1), 10q (PTEN, FAS), 2p (DNMT3A), 11q (USP28), 9p (CAAP1), but no recurrent rearrangements were identified. However, expression of five genes involved in rearrangements (TMEM244, EHD1, MTMR2, RNF123 and TOX) was altered in all patients. Fifteen rearrangements detected in Sézary syndrome patients and SeAx resulted in an expression of new fusion transcripts, nine of them were in frame (EHD1-CAPN12, TMEM66-BAIAP2, MBD4-PTPRC, PTPRC-CPN2, MYB-MBNL1, TFG-GPR128, MAP4K3-FIGLA, DCP1A-CCL27, MBNL1-KIAA2018) and five resulted in ectopic expression of fragments of genes not expressed in normal T-cells (BAIAP2, CPN2, GPR128, CAPN12, FIGLA). Our results not only underscored the genomic complexity of the Sézary cancer cell genome but also showed an unpreceded large variety of novel gene rearrangements resulting in fusions transcripts and ectopically expressed genes.

Hypermethylation of FOXP3 Promoter and Premature Aging of the Immune System in Female Patients with Panic Disorder?

Immunological abnormalities associated with pathological conditions, such as higher infection rates, inflammatory diseases, cancer or cardiovascular events are common in patients with panic disorder. In the present study, T cell receptor excision circles (TRECs), Forkhead-Box-Protein P3 gene (FOXP3) methylation of regulatory T cells (Tregs) and relative telomere lengths (RTLs) were investigated in a total and subsamples of 131 patients with panic disorder as compared to 131 age- and sex-matched healthy controls in order to test for a potential dysfunction and premature aging of the immune system in anxiety disorders. Significantly lower TRECs (p = 0.004) as well as significant hypermethylation of the FOXP3 promoter region (p = 0.005) were observed in female (but not in male) patients with panic disorder as compared to healthy controls. No difference in relative telomere length was discerned between patients and controls, but significantly shorter telomeres in females, smokers and older persons within the patient group. The presently observed reduced TRECs in panic disorder patients and FOXP3 hypermethylation in female patients with panic disorder potentially reflect impaired thymus and immunosuppressive Treg function, which might partly account for the known increased morbidity and mortality of anxiety disorders conferred by e.g. cancer and cardiovascular disorders.

In Vivo Silencing of A20 via TLR9-Mediated Targeted SiRNA Delivery Potentiates Antitumor Immune Response.

A20 is an ubiquitin-editing enzyme that ensures the transient nature of inflammatory signaling pathways induced by cytokines like TNF-α and IL-1 or pathogens via Toll-like receptor (TLR) pathways. It has been identified as a negative regulator of dendritic cell (DC) maturation and attenuator of their immunostimulatory properties. Ex vivo A20-depleted dendritic cells showed enhanced expression of pro-inflammatory cytokines and costimulatory molecules, which resulted in hyperactivation of tumor-infiltrating T lymphocytes and inhibition of regulatory T cells. In the present study, we demonstrate that a synthetic molecule consisting of a CpG oligonucleotide TLR9 agonist linked to A20-specific siRNAs silences its expression in TLR9+ mouse dendritic cells in vitro and in vivo. In the B16 mouse melanoma tumor model, silencing of A20 enhances the CpG-triggered induction of NFκB activity followed by elevated expression of IL-6, TNF-α and IL-12. This leads to potentiated antitumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. Our findings confirm the central role of A20 in controlling the immunostimulatory potency of DCs and provide a strategy for simultaneous A20 silencing and TLR activation in vivo.

Submicroscopic genomic rearrangements change gene expression in T-cell large granular lymphocyte leukemia.

OBJECTIVES: To better understand the molecular pathogenesis of T-cell large granular lymphocyte leukemia (T-LGL), we decided to search for those genetic alterations in T-LGL patients and MOTN-1 cell line (established from T-LGL patient) that have an impact on gene expression and as a result can influence cell biology. METHODS: Multicolor fluorescence in situ hybridization (mFISH) analysis of the MOTN-1 cell line was performed as well as paired-end next-generation sequencing (NGS; Illumina HiSeq2000) of this cell line and one T-LGL patient. In addition, chosen 6q region was characterized in three T-LGL patients using high-resolution comparative genomic hybridization (FT-CGH) and LM-PCR. Gene expression was studied by RNA sequencing (RNAseq; SOLID5500). RESULTS: Rearrangements were detected within 1p and 2q in MOTN-1 affecting expression of FGR, ZEB2, and CASP8, and within 6q in MOTN-1 and one T-LGL patient affecting MAP3K5 and IFNGR1. Nineteen genes, among them FOXN3, RIN3, AKT1, PPP2R5C, were overexpressed as a result of an amplification in 14q in one T-LGL patient. Two novel fusion transcripts were identified: CASP8-ERBB4 in MOTN-1 and SBF1-PKHD1L1 in T-LGL patient. CONCLUSIONS: This study showed that submicroscopic genomic rearrangements change gene expression in T-LGL. Several genes involved in rearrangements were previously linked to cancer and survival pattern that characterizes T-LGL cells.

The TCR γδ repertoire and relative gene expression characteristics of T-ALL cases with biclonal malignant Vδ1 and Vδ2 T cells.

Despite significant improvement in our understanding of T-cell acute lymphoblastic leukemia (T-ALL) biology and pathogenesis, many questions remain unanswered. In previous studies, we found a T-ALL case with two malignant T-cell clones with Vδ1Dδ2Dδ3Jδ1 and Vδ2Dδ3Jδ2 rearrangements. In this study, we further characterized T-ALL cases with two malignant clones containing Vδ1Dδ3Jδ1 and Vδ2Dδ1Jδ1 rearrangements using fine-tiling array comparative genomic hybridization, ligation-mediated polymerase chain reaction (LM-PCR), sequencing, and reverse transcription polymerase chain reaction (RT-PCR) analysis. We further analyzed the distribution and clonality of the T-cell receptor (TCR) Vγ and Vδ subfamily T cells in the two T-ALL cases by RT-PCR and GeneScan. Monoclonal Vδ1 and Vδ2 subfamilies were confirmed in both samples, the Vδ3 through Vδ7 subfamilies could not be detected in the T-ALL samples, whereas the oligoclonal Vδ8 subfamily could be identified. Based on the clinical finding that both of the T-ALL cases with two malignant T-cell clones had a poor outcome, we attempted to compare the expression pattern of genes related to T-cell activation and proliferation between cases with the malignant Vδ1 and Vδ2 T-cell clones and T-ALL cases with a mono-malignant Vα T-cell clone. We selected two T-ALL cases with VαJα rearrangements and analyzed the expression level of Notch1, TAL1, and the CARMA-BCL10-MALT-A20-NF-κB pathway genes by real-time PCR. A20 had significantly higher expression in the biclonal compared with the monoclonal T-ALL group (p=0.0354), and there was a trend toward higher expression for the other genes in the biclonal group with the exception of TAL1, although the differences were not statistically significant. In conclusion, we identified two T-ALL cases with biclonal malignant T-cell clones and described the characteristics of the biclonal T-ALL subtype and its gene expression pattern. Thus, our findings may improve the understanding of biclonal T-ALL.