Spotlight on hTERT Complex Regulation in Cutaneous T-Cell Lymphomas.

As a major cancer hallmark, there is a sustained interest in understanding the telomerase contribution to carcinogenesis in order to therapeutically target this enzyme. This is particularly relevant in primary cutaneous T-cell lymphomas (CTCL), a malignancy showing telomerase dysregulation with few investigative data available. In CTCL, we examined the mechanisms involved in telomerase transcriptional activation and activity regulation. We analyzed 94 CTCL patients from a Franco-Portuguese cohort, as well as 8 cell lines, in comparison to 101 healthy controls. Our results showed that not only polymorphisms (SNPs) located at the promoter of human telomerase reverse transcriptase (hTERT) gene (rs2735940 and rs2853672) but also an SNP located within the coding region (rs2853676) could influence CTCL occurrence. Furthermore, our results sustained that the post-transcriptional regulation of hTERT contributes to CTCL lymphomagenesis. Indeed, CTCL cells present a different pattern of hTERT spliced transcripts distribution from the controls, mostly marked by an increase in the hTERT ß+ variants proportion. This increase seems to be associated with CTCL development and progression. Through hTERT splicing transcriptome modulation with shRNAs, we observed that the decrease in the α-ß+ transcript induced a decrease in the cell proliferation and tumorigenic capacities of T-MF cells in vitro. Taken together, our data highlight the major role of post-transcriptional mechanisms regulating telomerase non canonical functions in CTCL and suggest a new potential role for the α-ß+ hTERT transcript variant.

New 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazoline and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinoline Derivatives: Synthesis and Biological Evaluation as Novel Anticancer Agents by Targeting G-Quadruplex.

The syntheses of novel 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazolines 12 and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinolines 13 are reported here in six steps starting from various halogeno-quinazoline-2,4-(1H,3H)-diones or substituted anilines. The antiproliferative activities of the products were determined in vitro against a panel of breast (MCF-7 and MDA-MB-231), human adherent cervical (HeLa and SiHa), and ovarian (A2780) cell lines. Disubstituted 6- and 7-phenyl-bis(3-dimethylaminopropyl)aminomethylphenyl-quinazolines 12b, 12f, and 12i displayed the most interesting antiproliferative activities against six human cancer cell lines. In the series of quinoline derivatives, 6-phenyl-bis(3-dimethylaminopropyl)aminomethylphenylquinoline 13a proved to be the most active. G-quadruplexes (G4) stacked non-canonical nucleic acid structures found in specific G-rich DNA, or RNA sequences in the human genome are considered as potential targets for the development of anticancer agents. Then, as small aza-organic heterocyclic derivatives are well known to target and stabilize G4 structures, their ability to bind G4 structures have been determined through FRET melting, circular dichroism, and native mass spectrometry assays. Finally, telomerase inhibition ability has been also assessed using the MCF-7 cell line.

Cutaneous Lymphocyte Antigen Is a Potential Therapeutic Target in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) such as Sézary syndrome or mycosis fungoides corresponds to an abnormal infiltration of T lymphocytes in the skin. CTCL cells have a heterogeneous phenotype and express cell adhesion molecules such as cutaneous lymphocyte antigen (CLA) supporting skin homing. The use of a mAb (HECA-452) against CLA significantly decreased transendothelial migration and survival of CTCL cells from patient samples and My-La cell line. The decrease of CLA expression by inhibition of its maturation enzyme, ST3 ß-galactoside α-2,3-sialyltransferase 4, also impaired CTCL cell migration, proliferation, and survival. We confirmed in vivo that treatment with anti-CLA mAb decreased the tumorigenicity as well as dissemination of CTCL cells in different tissues compared with the control group. Our findings provide evidence of the involvement of CLA in CTCL cell migration and survival, supporting that CLA inhibition could represent an actionable therapy in patients with CTCL.

Telomeric Repeat-Containing RNA (TERRA): A Review of the Literature and First Assessment in Cutaneous T-Cell Lymphomas.

Telomeric Repeat-containing RNA (TERRA) are long non-coding RNAs transcribed from telomeric DNA sequences from multiple chromosome ends. Major research efforts have been made to understand TERRA roles and functions in several physiological and pathological processes. We summarize herein available data regarding TERRA's roles in human cells and we report the first investigation in cutaneous T-cells lymphomas (CTCL) using real-time PCR. Among the TERRA analysed, our data suggest a particular role for TERRA 16p downregulation and TERRA 11q upregulation in CTCL lymphomagenesis.

Exploring hTERT promoter methylation in cutaneous T-cell lymphomas.

Cutaneous T-cell lymphomas (CTCLs) are telomerase-positive tumors expressing hTERT, although neither gene rearrangement/amplification nor promoter hotspot mutations could explain the hTERT re-expression. As the hTERT promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its re-expression. We analyzed hTERT promoter methylation status in CTCL cells compared with healthy cells. Gene-specific methylation analyses revealed a common methylation pattern exclusively in tumor cells. This methylation pattern encompassed a hypermethylated distal region from -650 to -150 bp and a hypomethylated proximal region from -150 to +150 bp. Interestingly, the hypermethylated region matches with the recently named TERT hypermethylated oncogenic region (THOR). THOR has been associated with telomerase reactivation in many cancers, but it has so far not been reported in cutaneous lymphomas. Additionally, we assessed the effect of THOR on two histone deacetylase inhibitors (HDACi), romidepsin and vorinostat, both approved for CTCL treatment and a DNA methyltransferase inhibitor (DNMTi) 5-azacytidine, unapproved for CTCL. Contrary to our expectations, the findings reported herein revealed that THOR methylation is relatively stable under these epigenetic drugs' pressure, whereas these drugs reduced the hTERT gene expression.

Targeting Epigenetic Modifiers Can Reduce the Clonogenic Capacities of Sézary Cells.

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) in which the human Telomerase Reverse Transcriptase (hTERT) gene is re-expressed. Current available treatments do not provide long-term response. We previously reported that Histone deacetylase inhibitors (HDACi, romidespin and vorinostat) and a DNA methyltransferase inhibitor (DNMTi, 5-azacytidine) can reduce hTERT expression without altering the methylation level of hTERT promoter. Romidepsin and vorinostat are approved for CTCL treatment, while 5-azacytidine is approved for the treatment of several hematological disorders, but not for CTCL. Here, using the soft agar assay, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells. Our data revealed that, besides hTERT downregulation, epidrugs' pressure reduced the proliferative and the tumor formation capacities in Sézary cells in vitro.

Reliable blood cancer cells' telomere length evaluation by qPCR.

BACKGROUND: Telomere shortening is linked to a range of different human diseases, hence reliable measurement methods are needed to uncover such associations. Among the plethora of telomere length measurement methods, qPCR is reported as easy to conduct and a cost-effective approach to study samples with low DNA amounts. METHODS: Cancer cells' telomere length was evaluated by relative and absolute qPCR methods. RESULTS: Robust and reproducible telomere length measurements were optimized taking into account a careful reference gene selection and by knowing the cancer cells ploidy. qPCR data were compared to "gold standard" measurement from terminal restriction fragment (TRF). CONCLUSIONS: Our study provides guidance and recommendations for accurate telomere length measurement by qPCR in cancer cells, taking advantage of our expertise in telomere homeostasis investigation in primary cutaneous T-cell lymphomas. Furthermore, our data emphasize the requirement of samples with both, high DNA quality and high tumor cells representation.

Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene.

Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.

Molecular alterations and tumor suppressive function of the DUSP22 (Dual Specificity Phosphatase 22) gene in peripheral T-cell lymphoma subtypes.

Monoallelic 6p25.3 rearrangements associated with DUSP22 (Dual Specificity Phosphatase 22) gene silencing have been reported in CD30+ peripheral T-cell lymphomas (PTCL), mostly with anaplastic morphology and of cutaneous origin. However, the mechanism of second allele silencing and the putative tumor suppressor function of DUSP22 have not been investigated so far. Here, we show that the presence, in most individuals, of an inactive paralog hampers genetic and epigenetic evaluation of the DUSP22 gene. Identification of DUSP22-specific single-nucleotide polymorphisms haplotypes and fluorescence in situ hybridization and epigenetic characterization of the paralog status led us to develop a comprehensive strategy enabling reliable identification of DUSP22 alterations. We showed that one cutaneous anaplastic large T-cell lymphomas (cALCL) case with monoallelic 6p25.3 rearrangement and DUSP22 silencing harbored exon 1 somatic mutations associated with second allele inactivation. Another cALCL case carried an intron 1 somatic splice site mutation with predicted deleterious exon skipping effect. Other tested PTCL cases with 6p25.3 rearrangement exhibited neither mutation nor deletion nor methylation accounting for silencing of the non-rearranged DUSP22 allele, thus inactivated by a so far unknown mechanism. We also characterized the expression status of four DUSP22 splice variants and found that they are all silenced in cALCL cases with 6p25.3 breakpoints. We finally showed that restoring expression of the physiologically predominant isoform in DUSP22-deficient malignant T cells inhibits cellular expansion by stimulating apoptosis and impairs soft agar clonogenicity and tumorigenicity. This study therefore shows that DUSP22 behaves as a tumor suppressor gene in PTCL.

Intrahepatic Xenograft of Cutaneous T-Cell Lymphoma Cell Lines: A Useful Model for Rapid Biological and Therapeutic Evaluation.

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of diseases primarily involving the skin that could have an aggressive course with circulating blood cells, especially in Sézary syndrome and transformed mycosis fungoides. So far, few CTCL cell lines have been adapted for in vivo experiments and their tumorigenicity has not been adequately assessed, hampering the use of a reproducible model for CTCL biological evaluation. In fact, both patient-derived xenografts and cell line xenografts at subcutaneous sites failed to provide a robust tool, because engraftment was dependent on mice strain and cell line subtype. Herein, we describe an original method of intrahepatic injection into adult NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice liver of both aggressive (My-La, HUT78, HH, MAC2A, and MAC2B) and indolent (FE-PD and MAC1) CTCL cell lines. Six of the seven CTCL cell lines were grafted with a high rate of success (80%). Moreover, this model provided a quick (15 days) and robust assay for in vivo evaluation of CTCL cell lines tumorigenicity and therapeutic response in preclinical studies. Such a reproducible model can be therefore used for further functional studies and in vivo drug testing.

Characterisation of inflammatory processes in Helicobacter pylori-induced gastric lymphomagenesis in a mouse model.

Gastric MALT lymphoma (GML) can be induced by Helicobacter pylori infection in BALB/c mice thymectomised at day 3 post-birth (d3Tx). This represented a unique opportunity to investigate the inflammatory process involved in the recruitment, proliferation and structuration of lymphoid infiltrates in the gastric mucosa of mice developing GML. Complementary molecular and proteomic approaches demonstrated that Th1 and Th2 cytokines were upregulated, along with activators/regulators of the lymphoid response and numerous chemokines. Interleukin-4, interferon γ, lymphotoxin-α and -ß were significantly upregulated and correlated with the inflammatory scores for all the d3Tx mice. GML lesions in d3Tx mice infected with H. pylori were associated with the presence of the inflammatory response. The dysregulation of numerous members of the tumour necrosis factor superfamily was also evident and suggests that they could play an important role in GML pathology, especially in light of their ability to promote and control lymphocyte proliferation.

Telomerase functions beyond telomere maintenance in primary cutaneous T-cell lymphoma.

Telomere erosion may be counteracted by telomerase. Here we explored telomere length (TL) and telomerase activity (TA) in primary cutaneous T-cell lymphoma (CTCL) by using quantitative polymerase chain reaction and interphase quantitative fluorescence in situ hybridization assays. Samples from patients with Sézary syndrome (SS), transformed mycosis fungoides (T-MF), and cutaneous anaplastic large cell lymphoma were studied in parallel with corresponding cell lines to evaluate the relevance of TL and TA as target candidates for diagnostic and therapeutic purposes. Compared with controls, short telomeres were observed in aggressive CTCL subtypes such as SS and T-MF and were restricted to neoplastic cells in SS. While no genomic alteration of the hTERT (human telomerase catalytic subunit) locus was observed in patients' tumor cells, TA was detected. To understand the role of telomerase in CTCL, we manipulated its expression in CTCL cell lines. Telomerase inhibition rapidly impeded in vitro cell proliferation and led to cell death, while telomerase overexpression stimulated in vitro proliferation and clonogenicity properties and favored tumor development in immunodeficient mice. Our data indicate that, besides maintenance of TL, telomerase exerts additional functions in CTCL. Therefore, targeting these functions might represent an attractive therapeutic strategy, especially in aggressive CTCL.

Reduced placental telomere length during pregnancies complicated by intrauterine growth restriction.

OBJECTIVES: Recent studies have shown that telomere length was significantly reduced in placentas collected at delivery from pregnancies complicated by intrauterine growth restriction secondary to placental insufficiency. Placental telomere length measurement during ongoing pregnancies complicated by intrauterine growth restriction has never been reported. This was the main objective of our study. METHODS: In our center, late chorionic villus samplings were performed between 18 and 37 weeks of amenorrhea in 24 subjects with severe intrauterine growth restriction (cases) and in 28 subjects with other indications for prenatal diagnosis (controls). Placental insufficiency was assessed by histo-pathological examination. Relative measurement of telomere length was carried out prospectively by quantitative Fluorescent In Situ Hybridization using fluorescent Peptide Nucleic Acid probes on interphase nuclei obtained from long-term cultured villi and with an automated epifluorescent microscope. A quantitative Polymerase Chain Reaction technique was performed to confirm the quantitative Fluorescent In Situ Hybridization results. The number of copies of gene loci encoding the RNA template (hTERC) and the catalytic subunit (hTERT) of the enzyme complex telomerase were also estimated in these placentas by Fluorescent In Situ Hybridization. RESULTS: Mean fluorescence intensity of telomere probes estimated by quantitative Fluorescent In Situ Hybridization was significantly less for cases compared to controls (p<0.001). This result indicated that mean telomere length was significantly reduced in placentas during pregnancies complicated by intrauterine growth restriction. Reduced telomere length was confirmed by the quantitative Polymerase Chain Reaction technique. No copy number variation of the hTERC and hTERT loci was noticed for cases, or for controls. CONCLUSION: This study clearly demonstrates a reduction of placental telomere length in ongoing pregnancies (from 18 to 37 weeks of amenorrhea) complicated by severe intrauterine growth restriction secondary to placental insufficiency.

CDKN2A-CDKN2B deletion defines an aggressive subset of cutaneous T-cell lymphoma.

Inactivation of the CDKN2A-CDKN2B locus has been reported in the most frequent subtypes of cutaneous T-cell lymphomas (CTCLs), mycosis fungoides, Sézary syndrome (SS) and CD30+ cutaneous anaplastic large cell lymphoma. To investigate whether genetic or epigenetic inactivation of CDKN2A-CDKN2B is more specifically observed in certain CTCL subtypes with clinical impact, we used array-comparative genomic hybridization, quantitative PCR, interphase fluorescent in situ hybridization and methylation analyses of p14(ARF) p16(INK4A) and p15(INK4B) promoters. We studied 67 samples from 58 patients with either transformed mycosis fungoides (n=24), SS (n=16) or CD30+ cutaneous anaplastic large cell lymphoma (n=18). We observed combined CDKN2A-CDKN2B deletion in both transformed mycosis fungoides (n=17, 71%) and SS patients (n=7, 44%), but, surprisingly, in only one CD30+ cutaneous anaplastic large cell lymphoma case. Interphase fluorescent in situ hybridization showed 9p21 loss in 17 out of 19 cases, with 9p21 deletion indicating either hemizygous (n=4) or homozygous (n=2) deletion, with mixed patterns in most patients (n=11). The limited size of 9p21 deletion was found to account for false-negative detection by either BAC arrays (n=9) or fluorescent in situ hybridization (n=2), especially in patients with Sézary syndrome (n=6). Methylation was found to be restricted to the p15(INK4B) gene promoter in patients with or without 9p21 deletion and did not correlate with prognosis. In contrast, CDKN2A-CDKN2B genetic loss was strongly associated with a shorter survival in CTCL patients (P=0.002) and more specifically at 24 months in transformed mycosis fungoides and SS patients (P=0.02). As immunohistochemistry for p16(INK4A) protein was not found to be informative, the genetic status of the CDKN2A-CDKN2B locus would be relevant in assessing patients with epidermotropic CTCLs in order to identify those cases where the disease was more aggressive.

Inactivation of p16INK4a/CDKN2A gene may be a diagnostic feature of large B cell lymphoma leg type among cutaneous B cell lymphomas.

The World Health Organization-European Organization for Research and Treatment of Cancer has individualized three main categories among the primary cutaneous B cell lymphoma (PCBCL): leg-type primary cutaneous large B cell lymphoma (PCLBCL leg type), primary cutaneous follicle center lymphoma (PCFCL), and primary cutaneous marginal zone lymphoma (PCMZL). The genetic features of 21 PCBCL cases (six PCLBCL leg type four PCFCL large cells, seven PCFCL small cells, and four PCMZL) were investigated by comparative genomic hybridization (CGH array). Fluorescent in situ hybridization (FISH) analysis was performed to confirm CGH array data and to detect lymphoma-associated gene rearrangements. p14(ARF)/p16(INK4a) CDKN2A gene quantification, methylation analysis, and immunohistochemical detection were also performed. CGH array showed a higher number of recurrent genetic imbalances in PCLBCL leg type (mean 62) than in PCFCL large cells (mean 34). PCFCL small cells and PCMZL exhibited fewer chromosomal alterations (mean 24 and 9). FISH analysis provided concordant results with CGH array data in 97% (98 of 101) assays and demonstrated a t(8;14)(q24;q32) in two of six PCLBCL leg type. Recurrent deletions in 9p21 (p14(ARF)/p16(INK4a)CDKN2A) were a constant finding in PCLBCL leg type (six of six). Conversely, PCFCL large cells exhibited recurrent 1p36 deletions (four of four) without deletion in 9p21 (zero of four). The diagnostic and prognostic impact of the p16(INK4a)CDKN2A gene status in PCBCL should therefore be confirmed on a larger series.