A mutation in a functional Sp1 binding site of the telomerase RNA gene (hTERC) promoter in a patient with Paroxysmal Nocturnal Haemoglobinuria

BACKGROUND: Mutations in the gene coding for the RNA component of telomerase, hTERC, have been found in autosomal dominant dyskeratosis congenita (DC) and aplastic anemia. Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal blood disorder associated with aplastic anemia and characterized by the presence of one or more clones of blood cells lacking glycosylphosphatidylinositol (GPI) anchored proteins due to a somatic mutation in the PIGA gene. METHODS: We searched for mutations in DNA extracted from PNH patients by amplification of the hTERC gene and denaturing high performance liquid chromatography (dHPLC). After a mutation was found in a potential transcription factor binding site in one patient electrophoretic mobility shift assays were used to detect binding of transcription factors to that site. The effect of the mutation on the function of the promoter was tested by transient transfection constructs in which the promoter is used to drive a reporter gene. RESULTS: Here we report the finding of a novel promoter mutation (-99C->G) in the hTERC gene in a patient with PNH. The mutation disrupts an Sp1 binding site and destroys its ability to bind Sp1. Transient transfection assays show that mutations in this hTERC site including C-99G cause either up- or down-regulation of promoter activity and suggest that the site regulates core promoter activity in a context dependent manner in cancer cells. CONCLUSIONS: These data are the first report of an hTERC promoter mutation from a patient sample which can modulate core promoter activity in vitro, raising the possibility that the mutation may affect the transcription of the gene in hematopoietic stem cells in vivo, and that dysregulation of telomerase may play a role in the development of bone marrow failure and the evolution of PNH clones

Telomere and telomerase in stem cells

Telomeres, guanine-rich tandem DNA repeats of the chromosomal end, provide chromosomal stability, and cellular replication causes their loss. In somatic cells, the activity of telomerase, a reverse transcriptase that can elongate telomeric repeats, is usually diminished after birth so that the telomere length is gradually shortened with cell divisions, and triggers cellular senescence. In embryonic stem cells, telomerase is activated and maintains telomere length and cellular immortality; however, the level of telomerase activity is low or absent in the majority of stem cells regardless of their proliferative capacity. Thus, even in stem cells, except for embryonal stem cells and cancer stem cells, telomere shortening occurs during replicative ageing, possibly at a slower rate than that in normal somatic cells. Recently, the importance of telomere maintenance in human stem cells has been highlighted by studies on dyskeratosis congenital, which is a genetic disorder in the human telomerase component. The regulation of telomere length and telomerase activity is a complex and dynamic process that is tightly linked to cell cycle regulation in human stem cells. Here we review the role of telomeres and telomerase in the function and capacity of the human stem cells

Nintedanib targets KIT D816V neoplastic cells derived from induced pluripotent stem cells of systemic mastocytosis

The KIT D816V mutation is found in >80% of patients with systemic mastocytosis (SM) and is key to neoplastic mast cell (MC) expansion and accumulation in affected organs. Therefore, KIT D816V represents a prime therapeutic target for SM. Here, we generated a panel of patient-specific KIT D816V induced pluripotent stem cells (iPSCs) from patients with aggressive SM and mast cell leukemia to develop a patient-specific SM disease model for mechanistic and drug-discovery studies. KIT D816V iPSCs differentiated into neoplastic hematopoietic progenitor cells and MCs with patient-specific phenotypic features, thereby reflecting the heterogeneity of the disease. CRISPR/Cas9n-engineered KIT D816V human embryonic stem cells (ESCs), when differentiated into hematopoietic cells, recapitulated the phenotype observed for KIT D816V iPSC hematopoiesis. KIT D816V causes constitutive activation of the KIT tyrosine kinase receptor, and we exploited our iPSCs and ESCs to investigate new tyrosine kinase inhibitors targeting KIT D816V. Our study identified nintedanib, a US Food and Drug Administration-approved angiokinase inhibitor that targets vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, as a novel KIT D816V inhibitor. Nintedanib selectively reduced the viability of iPSC-derived KIT D816V hematopoietic progenitor cells and MCs in the nanomolar range. Nintedanib was also active on primary samples of KIT D816V SM patients. Molecular docking studies show that nintedanib binds to the adenosine triphosphate binding pocket of inactive KIT D816V. Our results suggest nintedanib as a new drug candidate for KIT D816V-targeted therapy of advanced SM.Peer reviewe

Analysis of Marker-Defined HNSCC Subpopulations Reveals a Dynamic Regulation of Tumor Initiating Properties

Head and neck squamous carcinoma (HNSCC) tumors carry dismal long-term prognosis and the role of tumor initiating cells (TICs) in this cancer is unclear. We investigated in HNSCC xenografts whether specific tumor subpopulations contributed to tumor growth. We used a CFSE-based label retentions assay, CD49f (α6-integrin) surface levels and aldehyde dehydrogenase (ALDH) activity to profile HNSCC subpopulations. The tumorigenic potential of marker-positive and -negative subpopulations was tested in nude (Balb/c nu/nu) and NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice and chicken embryo chorioallantoic membrane (CAM) assays. Here we identified in HEp3, SQ20b and FaDu HNSCC xenografts a subpopulation of G0/G1-arrested slow-cycling CD49fhigh/ALDH1A1high/H3K4/K27me3low subpopulation (CD49f+) of tumor cells. A strikingly similar CD49fhigh/H3K27me3low subpopulation is also present in primary human HNSCC tumors and metastases. While only sorted CD49fhigh/ALDHhigh, label retaining cells (LRC) proliferated immediately in vivo, with time the CD49flow/ALDHlow, non-LRC (NLRC) tumor cell subpopulations were also able to regain tumorigenic capacity; this was linked to restoration of CD49fhigh/ALDHhigh, label retaining cells. In addition, CD49f is required for HEp3 cell tumorigenicity and to maintain low levels of H3K4/K27me3. CD49f+ cells also displayed reduced expression of the histone-lysine N-methyltransferase EZH2 and ERK1/2phosphorylation. This suggests that although transiently quiescent, their unique chromatin structure is poised for rapid transcriptional activation. CD49f− cells can “reprogram” and also achieve this state eventually. We propose that in HNSCC tumors, epigenetic mechanisms likely driven by CD49f signaling dynamically regulate HNSCC xenograft phenotypic heterogeneity. This allows multiple tumor cell subpopulations to drive tumor growth suggesting that their dynamic nature renders them a “moving target” and their eradication might require more persistent strategies

Characterisation of the Cell Line HC-AFW1 Derived from a Pediatric Hepatocellular Carcinoma

Current treatment of paediatric hepatocellular carcinoma (HCC) is often inefficient due to advanced disease at diagnosis and resistance to common drugs. The aim of this study was to generate a cell line derived from a paediatric HCC in order to expand research in this field. We established the HC-AFW1 cell line from a liver neoplasm of a 4-year-old boy through culturing of primary tumor specimens. The cell line has been stable for over one year of culturing and has a doubling time of 40 h. The tumour cells have an epithelial histology and express HCC-associated proteins such as Alpha-fetoprotein (AFP), Glypican 3, E-cadherin, CD10, CD326, HepPar1 and Vimentin. Forty-nine amino acids in exon 3 of β-Catenin that involve the phosphorylation sites of GSK3 were absent and β-Catenin is detectable in the cell nuclei. Cytogenetic analysis revealed large anomalies in the chromosomal map. Several alterations of gene copy numbers were detected by genome-wide SNP array. Among the different drugs tested, cisplatin and irinotecan showed effective inhibition of tumour cell growth in a proliferation assay at concentrations below 5 µg/ml. Subcutaneous xenotransplantation of HC-AFW1 cells into NOD/SCID mice resulted in fast growing dedifferentiated tumours with high levels of serum AFP. Histological analyses of the primary tumour and xenografts included national and international expert pathological review. Consensus reading characterised the primary tumour and the HC-AFW1-derived tumours as HCC. HC-AFW1 is the first cell line derived from a paediatric HCC without a background of viral hepatitis or cirrhosis and represents a valuable tool for investigating the biology of and therapeutic strategies for childhood HCC

Telomere shortening correlates with prognostic score at diagnosis and proceeds rapidly during progression of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is associated increased stem cell turnover. We have previously shown that short telomeres in chronic phase (CP) predict for early progression to blast phase ( BP). Poor prognostic score patients may therefore exhibit increased telomere loss at diagnosis and/or a greater than normal rate of loss during the disease course. We prospectively studied newly diagnosed CML patients for degree of telomere loss; measured telomere length in CML patients at all stages of disease; and performed follow-up sampling according to cytogenetic response to imatinib mesylate. Using flow-FISH, telomere length in peripheral blood leucocytes (PBL) from 32 consecutive newly diagnosed patients was measured ( with ex-vivo expanded T-cells as an internal BCR-ABL negative control), in addition to 65 samples from all CML stages and 7 paired CP/BP samples. Fifty-five normal individuals served as a control population. Patients who attained either a complete cytogenetic response (CCR, 0% Ph+, n = 10) or no CR (100% Ph+, n = 11) underwent follow-up measurement. All statistical tests were two sided. Telomeres in accelerated phase ( AP) and BP patients were significantly shorter than in CP, and mean telomere shortening was significantly greater in high-risk score than low-risk patients (P<0.05) at diagnosis. The rate of shortening during disease progression was 10 - 20 times the rate observed in normal granulocytes. BP samples had undergone at least 30 - 60 additional divisions from baseline Ph- telomere length. Our findings show that telomere shortening in CML is greatest in high-risk score patients at diagnosis, and occurs rapidly during disease progression