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Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer with limited therapeutic options for adult patients. Aurora kinases have drawn attention as potential targets in hematological neoplasms due to their high expression and biological functions. Aurora kinase A (AURKA) and AURKB are essential for a successful mitosis, acting in spindle mitotic organization and cytokinesis. Reversine is a synthetic purine analog that acts as a multi-kinase inhibitor with anti-neoplastic activity by targeting AURKA and AURKB. ALL patient gene expression data were retrieved from the Amazonia! database. For functional assays, Jurkat (T-ALL) and Namalwa (B-ALL) cells were exposed to increasing concentrations of reversine and submitted to various cellular and molecular assays. We found that AURKB expression was higher in ALL patient samples compared to normal lymphocytes (p

BACKGROUND Telomeres are specialized DNA structures that are critical to maintain cell homeostasis and to avoid genomic instability. Epidemiological studies have examined the association between leukocyte telomere length (LTL) and risk of cancers, but the findings remain conflicting. METHODS Mean LTL was measured by quantitative PCR in 97 patients with head and neck cancer (HNC) and 262 healthy controls. The association between LTL and patients' clinical status, such as smoke, alcoholism, and overall survival, were also evaluated. RESULTS The age-adjusted LTL was significantly shorter in patients with HNC in comparison to healthy controls (P = .0003). Patients with shortest LTL had an increased risk to develop HNC (P

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm with poor prognosis. Acquired telomerase reverse transcriptase gene promoter (TERTp) mutations are among the most frequent somatic non-coding mutations in cancers. In this study, the prevalence of TERTp mutations in 24 MCL and 21 other lymphoid neoplasias (oLN) was investigated. Eight MCL samples (33%) carried TERTp mutations, two homozygous and six heterozygous (seven C228T and one C250T), which directly correlated with higher TERT transcription, mitochondrial DNA copy number, and IGHV mutational status in MCL neoplastic cells. TERTp mutations were not found in oLN. TERTp mutations correlated with more lymphoma proliferation and tumor burden, as suggested by the higher number of lymphoma cells circulating in peripheral blood, and tended to associate with longer MCL telomeres, especially in homozygous mutants, although not statistically significant. Telomere-biology genes were overexpressed in MCL cells in comparison to healthy lymphocytes, but were not influenced by mutation status. The findings described for the first time that acquired TERTp mutations are common in MCL but not in other lymphoid neoplasms. It was also demonstrated that TERTp mutations are associated with higher TERT mRNA expression in MCL cells in vivo and higher tumor burden, suggesting these mutations as a driver event in MCL development and progression.

Telomeropathies are a group of phenotypically heterogeneous diseases molecularly unified by pathogenic mutations in telomere-maintenance genes causing critically short telomeres. X-linked dyskeratosis congenita (DC), the prototypical telomere disease, manifested with ectodermal dysplasia, cancer predisposition, and severe bone marrow failure, is caused by mutations in DKC1, encoding a protein responsible for telomerase holoenzyme complex stability. To investigate the effects of pathogenic DKC1 mutations on telomere repair and hematopoietic development, we derived induced pluripotent stem cells (iPSCs) from fibroblasts of a DC patient carrying the most frequent mutation: DKC1 p.A353V. Telomeres eroded immediately after reprogramming in DKC1-mutant iPSCs but stabilized in later passages. The telomerase activity of mutant iPSCs was comparable to that observed in human embryonic stem cells, and no evidence of alternative lengthening of telomere pathways was detected. Hematopoietic differentiation was carried out in DKC1-mutant iPSC clones that resulted in increased capacity to generate hematopoietic colony-forming units compared to controls. Our study indicates that telomerase-dependent telomere maintenance is defective in pluripotent stem cells harboring DKC1 mutation and unable to elongate telomeres, but sufficient to maintain cell proliferation and self-renewal, as well as to support the primitive hematopoiesis, the program that is recapitulated with our differentiation protocol. Keywords: Induced pluripotent stem cells (iPSCs), Telomeres, Telomerase, X-linked dyskeratosis congenita, DKC1, Hematopoietic differentiation

JAK2/STAT signaling participates in the Ph-negative myeloproliferative neoplasms (MPN) pathophysiology and has been targeted by ruxolitinib, a JAK1/2 inhibitor. In the present study, the impact of ruxolitinib treatment on cytoskeleton-related genes expression was explored. In SET2 cells, AURKA and AURKB expression/activity were downregulated in a dose- and time-dependent manner by ruxolitinib. Reversine, a multikinase inhibitor selective for aurora kinases, reduced cell viability in a dose- and/or time-dependent manner in JAK2V617F cells. Reversine significantly increased apoptosis and mitotic catastrophe, and reduced cell proliferation and clonogenic capacity in SET2 and HEL cells. In the molecular scenario, reversine induced DNA damage and apoptosis markers, as well as, reduced AURKA and AURKB expression/activity. In SET2 cells, reversine modulated the expression of 32 out of 84 apoptosis-related genes investigated, including downregulation of antiapoptotic (BCL2, BCL2L1, and BIRC5) and upregulation of proapoptotic (BIK, BINP3, and BNIP3L) genes. Synergism experiments indicated that low dose of reversine had a potentiating effect under ruxolitinib treatment at low doses in SET2 cells. In summary, our exploratory study establishes new targets, related to the regulation of the cellular cytoskeleton, for potential pharmacological intervention in MPN. These findings indicate that AURKA and AURKB participate in the JAK2/STAT signaling pathway and contribute to the MPN phenotype.

Background & Aims Short telomeres and genetic telomerase defects are risk factors for some human liver diseases, ranging from non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) to cirrhosis. In murine models, telomere dysfunction has been shown to metabolically compromise hematopoietic cells, liver, and heart via the activation of the p53-PGC axis Methods Tert- and Terc-deficient mice were challenged with liquid high fat diet (HFD). Liver metabolic contents were analyzed by CE-TOFMS and liver fat content was confirmed by confocal and electronic microscopy Results Tert-deficient but not Terc-deficient mice develop hepatocyte injury and frank steatosis when challenged with liquid HFD. Upon HFD, Tert-/- hepatocytes fail to engage the citric acid cycle (TCA), with an imbalance of NADPH/NADP+ and NADH/NAD+ ratios and depletion of intermediates of TCA cycle, such as cis-aconitic acid. Telomerase deficiency caused an intrinsic metabolic defect unresponsive to environmental challenge. Chemical inhibition of telomerase by zidovudine recapitulated the abnormal Tert-/- metabolic phenotype in Terc-/- hepatocytes Conclusions Our findings indicate that in telomeropathies short telomeres are not the only molecular trigger and telomerase enzyme deficiency provokes hepatocyte metabolic dysfunction, abrogates response to environmental challenge, and causes cellular injury and steatosis, providing a mechanism for liver damage in telomere diseases. This article is protected by copyright. All rights reserved.

Telomeres are repetitive DNA sequences at linear chromosome termini, protecting chromosomes against end-to-end fusion and damage, providing chromosomal stability. Telomeres shorten with mitotic cellular division, but are maintained in cells with high proliferative capacity by telomerase. Loss-of-function mutations in telomere-maintenance genes are genetic risk factors for cirrhosis development in humans and murine models. Telomerase deficiency provokes accelerated telomere shortening and dysfunction, facilitating genomic instability and oncogenesis. Here we examined whether telomerase mutations and telomere shortening were associated with hepatocellular carcinoma (HCC) secondary to cirrhosis. Telomere length of peripheral blood leukocytes was measured by Southern blot and qPCR in 120 patients with HCC associated with cirrhosis and 261 healthy subjects. HCC patients were screened for telomerase gene variants (in TERT and TERC) by Sanger sequencing. Age-adjusted telomere length was comparable between HCC patients and healthy subjects by both Southern blot and qPCR. Four non-synonymous TERT heterozygous variants were identified in four unrelated patients, resulting in a significantly higher mutation carrier frequency (3.3%) in patients as compared to controls (p = 0.02). Three of the four variants (T726M, A1062T, and V1090M) were previously observed in patients with other telomere diseases (severe aplastic anemia, acute myeloid leukemia, and cirrhosis). A novel TERT variant, A243V, was identified in a 65-year-old male with advanced HCC and cirrhosis secondary to chronic hepatitis C virus (HCV) and alcohol ingestion, but direct assay measurements in vitro did not detect modulation of telomerase enzymatic activity or processivity. In summary, constitutional variants resulting in amino acid changes in the telomerase reverse transcriptase were found in a small proportion of patients with cirrhosis-associated HCC.

Dengue is the most important arbovirus in the world with an estimated of 50 million dengue infections occurring annually and approximately 2.5 billion people living in dengue endemic countries. Yellow fever is a viral hemorrhagic fever with high mortality that is transmitted by mosquitoes. Effective vaccines against yellow fever have been available for almost 70 years and are responsible for a significant reduction of occurrences of the disease worldwide; however, approximately 200,000 cases of yellow fever still occur annually, principally in Africa. Therefore, it is a public health priority to develop antiviral agents for treatment of these virus infections. Crotalus durissus terrificus snake, a South American rattlesnake, presents venom with several biologically actives molecules. In this study, we evaluated the antiviral activity of crude venom and isolated toxins from Crotalus durissus terrificus and found that phospholipases A₂ showed a high inhibition of Yellow fever and dengue viruses in VERO E6 cells.

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm with poor prognosis. Acquired telomerase reverse transcriptase gene promoter (TERTp) mutations are among the most frequent somatic non-coding mutations in cancers. In this study, the prevalence of TERTp mutations in 24 MCL and 21 other lymphoid neoplasias (oLN) was investigated. Eight MCL samples (33%) carried TERTp mutations, two homozygous and six heterozygous (seven C228T and one C250T), which directly correlated with higher TERT transcription, mitochondrial DNA copy number, and IGHV mutational status in MCL neoplastic cells. TERTp mutations were not found in oLN. TERTp mutations correlated with more lymphoma proliferation and tumor burden, as suggested by the higher number of lymphoma cells circulating in peripheral blood, and tended to associate with longer MCL telomeres, especially in homozygous mutants, although not statistically significant. Telomere-biology genes were overexpressed in MCL cells in comparison to healthy lymphocytes, but were not influenced by mutation status. The findings described for the first time that acquired TERTp mutations are common in MCL but not in other lymphoid neoplasms. It was also demonstrated that TERTp mutations are associated with higher TERT mRNA expression in MCL cells in vivo and higher tumor burden, suggesting these mutations as a driver event in MCL development and progression.

Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R(2) = 0.60; p

Excessive telomere erosion is the molecular etiology of a group of disorders (dyskeratosis congenita, aplastic anemia, idiopathic pulmonary fibrosis) collectively called telomeropathies. Telomere length measurement is an essential diagnostic test for these diseases. The most commonly used methods are terminal restriction fragment (TRF) analysis by Southern blotting (the gold-standard method), flow cytometry combined with fluorescence in situ hybridization (flow-FISH), and quantitative PCR (qPCR). Although the clinical use of these methods has been reported, their utility and characteristics have not been widely compared. Measurement techniques and coefficients of variations often differ among diagnostic services. Here, we directly compared the accuracy, reproducibility, sensitivity, and specificity of flow-FISH and qPCR in comparison to TRF to measure peripheral blood leukocyte’s telomere length in healthy individuals and patients with telomeropathies. TRF analyses and flow-FISH showed good correlation in the analysis of samples from healthy subjects (R2=0.60; p Disclosures No relevant conflicts of interest to declare.

Background Myelodysplastic syndromes (MDS) comprise a group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis and propensity for progression to acute myeloid leukemia. Acquired mutations in the gene encoding RNA splicing factor 3B subunit 1 ( SF3B1 ) are highly associated with the MDS subtypes presenting ring sideroblasts, and represent a specific nosological entity. The effects of these mutations on clinical outcomes are diverse and contrasting. Methods A cohort of 91 Brazilian MDS patients, including patients with ring sideroblasts in the bone marrow, were screened for mutations in the SF3B1 hotspots (exons 12–15) by direct Sanger sequencing. Results SF3B1 heterozygous mutations were identified in six patients (7%), all of them with ring sideroblasts, thus confirming the association between SF3B1 mutations and myelodysplastic syndrome subtypes bearing this morphologic feature (frequency of 6/13, p -value Conclusion This is the first screening of SF3B1 mutations in a cohort of Brazilian myelodysplastic syndrome patients. Our findings confirm that mutations in this splicing gene correlate with bone marrow ringed sideroblasts.

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome known as the prototype of telomere diseases. In addition to the clinical triad (nail dystrophy, hyperpigmentation, and leukoplakia), very short telomeres (below the 1st percentile) is a marker for the diagnosis of DC (Calado & Young, NEJM 2009). Telomere dysfunction was associated with DC after discovery of DKC1 mutations in patients presenting the X-linked form of the syndrome. Novel mutations in telomere biology genes (TERC, TERT, NOP10, NHP2, TINF2, TCAB1, CTC1, RTEL1, ACD, and PARN) have been described in patients with DC. Variations in all these genes can affect telomere protection and maintenance, leading to telomere shortening and development of telomeropathies. In this study, we mapped the TERT, TERC, DKC1, and TINF2 genes for mutations in 15 patients (median age = 10 years; M/F = 11/4) with DC and very short telomeres, in order to classify these cases in a molecular level and determine the frequency of these mutations in our cohort. Survival for twelve patients who underwent allogeneic hematopoietic stem cell transplant (HSCT) was assessed and correlated with mutational status and telomere length. Diagnosis of DC was made according to the definition of Calado & Young (2009). Telomere length was measured in nucleated blood cells by flow-FISH and mutational screening was performed on genomic DNA extracted from peripheral blood cells by direct sequencing. Seven non-synonymous mutations were identified in TINF2 (40%), two in DKC1 (13%), one in TERT (6%), and one in TERC (6%). The TINF2 variants R282H and R282C had been already described as pathogenic, as well T66A and A353V DKC1 variants (Knight et al, 1999; Savage et al, 2008; Walne et al, 2008). The heterozygous variant R282H (c. 845 G>A) in TINF2 were found in 4 unrelated patients. One of them also harbor the variants Q120R and Q157H in the same gene. The heterozygous mutation in TINF2 R282C (c. 844 C>T) was found in one patient, that also presented the common polymorphism A279T in TERT. The pathogenic variants T66A (c.196A>G) and A353V (c.1058C>T) in DKC1 were found in two different male patients. Moreover, three novel mutations were identified in our cohort, r.94 C>T in TERC, F290C in TINF2, and R696Cin TERT. The heterozygous mutation r.94 (C>T) found in TERC was located at the pseudoknot P2b region of the gene and the patient who carries that presented a severe aplastic anemia and all DC clinical triad. The novel heterozygous F290C (c.859 T>G) variant is located at the "hot spot" in exon 6 of TINF2 andwas found in one patient that presented a severe phenotype of DC. In silico analysis with SIFT and Polyphen-2 predicted that this variant is not tolerated and probably damaging, which is consistent with the pathogenicity of the mutation. The homozygous mutation R696C (c.2086 C>T) in TERT was found in one patient and also in his two brothers. All of them presented reduced blood cell count, clinical features of DC, and severe aplastic anemia. The family screening identified the father and sister as heterozygous for the same mutation, but both asymptomatic. DNA sample from the mother were not available for this study. In silico analysis by SIFT and Polyphen 2.0, predicted that the R696C mutation is not tolerant and possible damaging to telomerase activity, respectively. To validate in silico analysis, TRAP assay with cell lysates obtained from telomerase-negative VA13 cell line transfected with wild type or R696C mutated TERT vector and TERC vector is under evaluation. Consistently with previously studies, telomere length in patients with TINF2 mutations were the shortest compared with the other telomeres genes mapped in this study. Although, the phenotype and severity of the disease does not appear to change according to the mutated gene. Also, the mutational status (p=0.28) or telomere length (p=0.21) did not influence the survival rates of patients after HSCT. Flow-FISH was able to identify patients with very short telomeres and validated telomere length measurement as a diagnostic tool for DC. Direct sequencing of the most commonly mutated genes in DC in a cohort of patients with telomeres below 1st percentile was able to characterize the genetic cause of this disease in more than 70% of the cases. The identification of genetic defect in DC can manage clinical decisions and is essential to genetic counseling prior to bone marrow transplantation. Disclosures No relevant conflicts of interest to declare.