[Show abstract][Hide abstract] ABSTRACT: Background:
The identification of oncogenic driver mutations has largely relied on the assumption that genes that exhibit more mutations than expected by chance are more likely to play an active role in tumorigenesis. Major cancer sequencing initiatives have therefore focused on recurrent mutations that are more likely to be drivers. However, in specific genetic contexts, low frequency mutations may also be capable of participating in oncogenic processes. Reliable strategies for identifying these rare or even patient-specific (private) mutations are needed in order to elucidate more personalized approaches to cancer diagnosis and treatment.
Here we performed whole-exome sequencing on three cases of childhood pre-B acute lymphoblastic leukemia (cALL), representing three cytogenetically-defined subgroups (high hyperdiploid, t(12;21) translocation, and cytogenetically normal). We applied a data reduction strategy to identify both common and rare/private somatic events with high functional potential. Top-ranked candidate mutations were subsequently validated at high sequencing depth on an independent platform and in vitro expression assays were performed to evaluate the impact of identified mutations on cell growth and survival.
We identified 6 putatively damaging non-synonymous somatic mutations among the three cALL patients. Three of these mutations were well-characterized common cALL mutations involved in constitutive activation of the mitogen-activated protein kinase pathway (FLT3 p.D835Y, NRAS p.G13D, BRAF p.G466A). The remaining three patient-specific mutations (ACD p.G223V, DOT1L p.V114F, HCFC1 p.Y103H) were novel mutations previously undescribed in public cancer databases. Cytotoxicity assays demonstrated a protective effect of the ACD p.G223V mutation against apoptosis in leukemia cells. ACD plays a key role in protecting telomeres and recruiting telomerase. Using a telomere restriction fragment assay, we also showed that this novel mutation in ACD leads to increased telomere length in leukemia cells.
This study identified ACD as a novel gene involved in cALL and points to a functional role for ACD in enhancing leukemia cell survival. These results highlight the importance of rare/private somatic mutations in understanding cALL etiology, even within well-characterized molecular subgroups.
BMC Cancer 09/2015; 15(1):621. DOI:10.1186/s12885-015-1639-5 · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The key step of carcinogenesis is the malignant transformation which is fundamentally a telomere biology dysfunction permitting cells to bypass the Hayflick limit and to divide indefinitely and uncontrollably. Thus all partners and structures involved in normal and abnormal telomere maintenance, protection and lengthening can be considered as potential anti-cancer therapeutic targets. In this Point of View, we discuss, highlight and provide new perspectives from the current knowledge and understanding to position the different aspects of telomere biology and dysfunction as diagnostic, preventive and curative tools in the field of cancer.
[Show abstract][Hide abstract] ABSTRACT: Maintenance of telomere length and structure is essential for cell survival. Telomere synthesis is mediated by the ribonucleoprotein telomerase in 90% of cancer cells, and is regulated mainly by transcription of the human telomerase reverse transcriptase subunit, hTERT. However, transcriptome analysis reveals complex splicing patterns and to date, twenty-two alternatively-spliced hTERT mRNAs have been reported, yet their functions have not been fully elucidated. The best characterized hTERT spliced variants encode for inactive proteins that possess specific deletions within the hTERT catalytic domains. We studied two less well characterized hTERT splice variants (termed INS3 and 4) that encode proteins with intact reverse transcriptase motifs, but alternative C-domains due to insertion of intronic sequences. We determined the prevalence of these mRNA variants in primary cells, telomerase-positive cells and in alternative lengthening of telomere (ALT) cells and found the transcripts to be expressed mainly in telomerase-positive cell lines and to be translated into proteins as illustrated by their association with polysomes. These variants were inactive when expressed in vitro or in cells, retained DNA substrate binding in vitro but were impaired in binding the telomerase RNA component when expressed in, and immunoprecipitated from either telomerase-positive or telomerase-negative ALT cells coexpressing the telomerase RNA component. Stable expression of INS3 and INS4 variants in a hepatocarcinoma cell line inhibited telomerase activity, shortened telomeres and slowed cell growth suggesting a potential dominant-negative function.
[Show abstract][Hide abstract] ABSTRACT: We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells.
PLoS ONE 07/2013; 8(7):e70428. DOI:10.1371/journal.pone.0070428 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in the dyskerin gene (DKC1) cause X-linked dyskeratosis congenita (DC), a rare and fatal premature aging syndrome characterized by defective telomere maintenance. Dyskerin is a highly conserved nucleolar protein, and a component of the human telomerase complex that is essential for human telomerase RNA (hTR) stability. However, its regulation remains poorly understood. Here, we report that dyskerin can be modified by small ubiquitin-like modifiers (SUMO). We find that human DC-causing mutations in highly conserved dyskerin SUMOylation consensus sites lead to impaired hTR accumulation, telomerase activity and telomere maintenance. Finally, we show that modification of dyskerin by SUMOylation is required for its stability. Our findings provide the first evidence that dyskerin stability is regulated by SUMOylation and that mutations altering dyskerin SUMOylation can lead to defects in telomere maintenance that are characteristic of DC.
Human Molecular Genetics 05/2013; 22(17). DOI:10.1093/hmg/ddt204 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Short repetitive G-rich telomeric sequences are synthesized by telomerase, a ribonucleoprotein consisting of the telomerase reverse transcriptase, TERT, and an integrally associated RNA. Human TERT (hTERT) can repetitively reverse transcribe its RNA template, acting processively to add multiple telomeric repeats onto the same substrate. We investigated if certain threshold levels of telomerase activity and processivity are required to maintain telomere function and immortalize human cells with limited lifespan. We assessed hTERT variants with mutations in motifs implicated in processivity and interaction with DNA, the 'Insertion in Fingers' domain (V791Y) and the E 'primer grip' motif (W930F). hTERT-W930F and hTERT-V791Y reconstitute reduced levels of DNA synthesis and processivity compared with wild-type telomerase. Interestingly hTERT-W930F is more defective in translocation than hTERT-V791Y. Nonetheless, hTERT-W930F, but not hTERT-V791Y, immortalizes limited lifespan human cells. Both hTERT-W930F- and hTERT-V791Y- expressing cells harbor short telomeres, measured as signal free ends (SFEs), yet SFEs persist only in hTERT-V791Y cells, which undergo apoptosis, likely as a consequence of a defect in recruitment of hTERT-V791Y to telomeres. Our study is the first to demonstrate that low levels of DNA synthesis on the order of 20% of wild-type telomerase levels, and extension of as few as three telomeric repeats are sufficient to maintain functional telomeres and immortalize limited lifespan human cells.
Molecular biology of the cell 02/2013; 24(9). DOI:10.1091/mbc.E12-12-0889 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Telomerase is a ribonucleoprotein consisting of a catalytic subunit, the telomerase reverse transcriptase, TERT, and an integrally associated RNA, TR, which contains a template for the synthesis of short repetitive G-rich DNA sequences at the ends of telomeres. Telomerase can repetitively reverse transcribe its short RNA template, acting processively to add multiple telomeric repeats onto the same DNA substrate. The contribution of enzyme processivity to telomere length regulation in human cells is not well characterized. In cancer cells, under homeostatic telomere length-maintenance conditions, telomerase acts processively, while under nonequilibrium conditions, telomerase acts distributively on the shortest telomeres. To investigate the role of increased telomerase processivity on telomere length regulation in human cells with limited lifespan that are dependent on human TERT (hTERT) for lifespan extension and immortalization, we mutated the leucine at position 866 in the reverse transcriptase C motif of hTERT to a tyrosine (L866Y), which is the amino acid found at a similar position in HIV-1 reverse transcriptase. We report that, similar to the previously reported 'gain of function' Tetrahymena telomerase mutant (L813Y), the human telomerase variant displays increased processivity. hTERT-L866Y, like wild-type hTERT can immortalize and extend the lifespan of limited lifespan cells. Moreover, hTERT-L866Y expressing cells display heterogenous telomere lengths, telomere elongation, multiple telomeric signals indicative of fragile sites and replicative stress, and an increase in short telomeres, which is accompanied by telomere trimming events. Our results suggest that telomere length and homeostasis in human cells may be regulated by telomerase enzyme processivity.
[Show abstract][Hide abstract] ABSTRACT: The minimal vertebrate telomerase enzyme is composed of a protein component (telomerase reverse transcriptase, TERT) and an RNA component (telomerase RNA, TR). Expression of these two subunits is sufficient to reconstitute telomerase activity in vitro, while the formation of a holoenzyme comprising telomerase-associated proteins is necessary for proper telomere length maintenance. Previous reports demonstrated the high processivity of the human telomerase complex and the interspecies compatibility of human TERT (hTERT). In this study, we tested the function of the only known viral telomerase RNA subunit (vTR) in association with human telomerase, both in a cell-free system and in human cells. When vTR is assembled with hTERT in a cell-free environment, it is able to interact with hTERT and to reconstitute telomerase activity. However, in human cells, vTR does not reconstitute telomerase activity and could not be detected in the human telomerase complex, suggesting that vTR is not able to interact properly with the proteins constituting the human telomerase holoenzyme.
[Show abstract][Hide abstract] ABSTRACT: This chapter contains sections titled: Conservation of Telomere Function and the Discovery of TelomeraseThe Discovery of the Two Minimal Telomerase ComponentsTelomerase Beyond the Minimal Components: Associated ProteinsRegulation of Telomerase by Telomeric Proteins and RNASTelomerase, Telomere Maintenance, Cancer, and AgingTelomerase Beyond Telomere SynthesisTelomere Maintenance Without TelomeraseConclusion
[Show abstract][Hide abstract] ABSTRACT: A rationally designed progression of phenanthroimidazole platinum(II) complexes were examined for their ability to target telomere-derived intramolecular G-quadruplex DNA. Through the use of circular dichroism, fluorescence displacement assays, and molecular modeling we show that these complexes template and stabilize G-quadruplexes from sequences based on the human telomeric repeat (TTAGGG)(n). The greatest stabilization was observed for the p-chlorophenyl derivative 6((G4)DC(50) =0.31 μM). We also show that the G-quadruplex binding complexes are able to inhibit telomerase activity through a modified telomerase repeat amplification protocol (TRAP-LIG assay). Preliminary cell studies show that complex 6 is preferentially cytotoxic toward cancer over normal cell lines, indicating its potential use in cancer therapy.
[Show abstract][Hide abstract] ABSTRACT: Telomere maintenance is essential for cellular immortality, and most cancer cells maintain their telomeres through the enzyme telomerase. Telomeres and telomerase represent promising anticancer targets. However, 15% of cancer cells maintain their telomeres through alternative recombination-based mechanisms, and previous analyses showed that recombination-based telomere maintenance can be activated after telomerase inhibition. We determined whether telomeric recombination can also be promoted by telomere dysfunction. We report for the first time that telomeric recombination can be induced in human telomerase-positive cancer cells with dysfunctional telomeres.
Molecular biology of the cell 01/2011; 22(2):179-88. DOI:10.1091/mbc.E10-02-0173 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Telomerase synthesizes telomeric sequences and is minimally composed of a reverse transcriptase (RT) known as TERT and an RNA known as TR. We reconstituted heterologous mouse (m) and human (h) TERT-TR complexes and chimeric mTERT-hTERT-hTR complexes in vitro and in immortalized human alternative lengthening of telomere (ALT) cells. Our data suggest that species-specific determinants of activity, processivity and telomere function map not only to the TR but also to the TERT component. The presence of hTERT-hTR, but not heterologous TERT-TR complexes or chimeric mTERT-hTERT-hTR complexes, significantly reduced the percentage of chromosomes without telomeric signals in ALT cells. Moreover, heterologous and chimeric complexes were defective in recruitment to telomeres. Our results suggest a requirement for several hTERT domains and interaction with multiple proteins for proper recruitment of telomerase to the shortest telomeres in human ALT cells. Late-passage mTERT(-/-) mouse embryonic stem (ES) cells ectopically expressing hTERT or mTERT harboured fewer chromosome ends without telomeric signals and end-to-end fusions than typically observed in late-passage mTERT(-/-) ES cells. The ability of hTERT to function at mouse telomeres and the inability of mTERT to function at human telomeres suggest that mechanisms regulating the recruitment and activity of hTERT at mouse telomeres might be less stringent than the mechanisms regulating mTERT at human telomeres.
[Show abstract][Hide abstract] ABSTRACT: Cellular viability requires telomere maintenance, which, in mammals, is mainly mediated by the reverse transcriptase telomerase. Telomerase core components are a catalytic subunit TERT and an RNA subunit TR (hTR in humans, mTR in mouse) that carries the template to generate telomeres de novo. Telomere dysfunction can lead to senescence or apoptosis and impairs the continued growth of immortal cancerous cell lines. The introduction of a template-mutated hTR in telomerase-positive and telomerase-negative human cell lines results in dramatic growth defects. No study has addressed the consequences of expressing a template-mutated mTR in mouse immortal cell lines. Therefore, we analyzed the effects of long-term expression of a template-mutated mTR in the telomerase-positive and telomerase-negative murine cell lines CB17 and DKO301, respectively. Whereas the CB17 clones expressing the template-mutated mTR did not demonstrate any growth impairment, many of the DKO301 clones expressing the template-mutated mTR underwent growth and cell cycle defects and eventual cell death. These results suggest that in the absence of wild-type telomerase, the expression of the template-mutated mTR likely perturbs telomere function, leading to decreased cellular viability. Furthermore, whereas the expression of template-mutated hTR in telomerase-negative human cell lines leads to immediate cellular toxicity, the expression of the template-mutated mTR in the telomerase-negative mouse cell line did not.
Cancer letters 03/2009; 275(2):266-76. DOI:10.1016/j.canlet.2008.10.027 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dyskeratosis congenita (DC) is a rare syndrome, characterized by cutaneous abnormalities and premature death caused by bone marrow failure. In this issue of Genes & Development, Hockemeyer and colleagues (pp. 1773-1785) report a new mouse model that reconstitutes key features of DC. Disease phenotypes are generated by a POT1b deletion in a telomerase-deficient background that accelerates the shortening of telomeres by degradation.
Genes & Development 08/2008; 22(13):1731-6. DOI:10.1101/gad.1695808 · 10.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this contribution, we report that a self-assembled platinum molecular square [Pt(en)(4,4'-dipyridyl)]4 can act as an efficient G-quadruplex binder and telomerase inhibitor. Molecular modeling studies show that the square arrangement of the four bipyridyl ligands, the highly electropositive nature of the overall complex, as well as hydrogen bonding interactions between the ethylenediamine ligands and phosphates of the DNA backbone all contribute to the observed strong binding affinity to the G-quadruplex. Through thermal denaturation studies with duplex and quadruplex FRET probes and enzymatic assays, we demonstrate that this platinum square strongly binds to G-quadruplexes and can act as an inhibitor of telomerase. This study thus shows the potential of supramolecular self-assembly to readily generate scaffolds of unique geometries for effective targeting of G-quadruplexes and for the ultimate development of selective antitumor therapies.
Journal of the American Chemical Society 08/2008; 130(31):10040-1. DOI:10.1021/ja8014023 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The indefinite growth of cancer cells requires telomere maintenance, which, in the majority of mammalian cancers is mediated via the enzyme telomerase. The core components of telomerase are a catalytic reverse transcriptase (hTERT in human, mTERT in mouse) and an RNA (TR) that contains the template for the replenishment of telomeres. Fundamental differences in human and mouse telomerase and telomere biology should be considered when using mouse models for the study of human cancers. The responses to telomerase inhibition by the expression of a catalytically-inactive dominant-negative mutant of hTERT (hTERT-DN) vary in human cells with different telomere lengths. Only one similar study has been performed in a mouse cell line with short telomeres (RenCa, 7 kb). Thus, we asked whether the responses to telomerase inhibition are also telomere-length dependent in mouse cells by analyzing long-term stable expression of mTERT-DN in the CB17 cell line (telomere length, 11 kb). A brief initial telomerase inhibition was insufficient to mediate telomere shortening and led to extremely rapid telomerase reactivation due to an increase in the level of expression of the endogenous mTERT. Thus, mouse cells, in contrast to human cells may not tolerate telomerase inhibition by introduction of mTERT-DN, independently of telomere length.
Experimental Cell Research 03/2008; 314(3):668-75. DOI:10.1016/j.yexcr.2007.10.020 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Telomeres serve to camouflage chromosome ends from the DNA damage response machinery. Telomerase activity is required to maintain telomeres. One consequence of telomere dysfunction is cellular senescence, a permanent growth arrest state. We review the key regulators of cellular senescence and recent in vivo evidence which supports p53-dependent senescence induced by short telomeres as a potent tumor suppressor pathway. The in vivo link between cellular senescence and tumor regression is also discussed. The relationship between short telomere length and ageing or disease states in various cells of the body is increasingly reported. Paradoxically, the introduction of telomerase is proposed as a method to combat ageing via cell therapy and a possible method to regenerate tissue, while telomerase inhibition and telomere shortening is suggested as a possible therapy to defeat cancers with intact p53. Researchers thus face the challenge of understanding the complex processes which regulate the potential benefits of both telomerase inhibition and activation.
Mechanisms of Ageing and Development 01/2008; 129(1-2):3-10. DOI:10.1016/j.mad.2007.11.007 · 3.40 Impact Factor