Cell Stem Cell

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Pushing the Limits: Defeating Leukemia Stem Cells by Depleting Telomerase Ya-Huei Kuo1,* and Ravi Bhatia1,* 1Division of Hematopoietic Stem Cell and Leukemia Research, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA *Correspondence: [email protected] (Y.-H.K.), [email protected] (R.B.) http://dx.doi.org/10.1016/j.stem.2014.11.014

Leukemia stem cells (LSCs), featuring unlimited self-renewal capacity and chemoresistance, are critical cellular targets for new treatments to improve outcomes for acute myeloid leukemia (AML). In this issue of Cell Stem Cell, Bruedigam et al. (2014) demonstrate that inhibition of telomerase is damaging to LSCs and may represent a promising therapeutic approach in AML. Telomeres, the nucleoprotein complexes with repetitive sequences that cap the ends of eukaryotic chromosomes, are essential for maintaining replicative capacity and genomic stability. The length of telomeres shortens with each replication cycle, thereby limiting cellular life span. Telomere maintenance is particularly important for pluripotent stem cells (Pucci et al., 2013) and multipotent stem cells, including hematopoietic stem cells (Wang et al., 2014), and is critical for tissue homeostasis or regeneration. The telomerase complex, composed of a catalytic subunit telomerase reverse transcriptase (TERT), a telomerase RNA component (TERC), and a shelterin scaffold can lengthen telomeric DNA through a RNA-directed DNA polymerization mechanism. Telomere shortening is among the hallmarks of aging and malignant cells. Once a telomere is critically short, the chromosome ends elicit a double-strand-breaklike DNA damage response that results in growth arrest and cell death. Ageadjusted telomere length appears to be significantly reduced in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and chronic

myeloid leukemia (CML) as compared to matched controls. Telomere shortening may reflect the increased replication required for leukemia development, but it could also result from altered telomere regulatory mechanisms. Indeed, telomerase mutations are associated with myeloid malignancies, especially MDS and AML (Townsley et al., 2014). Most cancers have relatively short telomeres but high levels of telomerase activity compared to normal cells. Enhanced telomerase activity likely represents an important adaptive mechanism that allows leukemia cells to continue to replicate despite marked telomeric shortening. Telomerase activity has been shown to be increased in CML and to be required for leukemia development (Vicente-Duen˜as et al., 2012). In addition, the AML-related fusion genes MLL-AF4 and AML1-ETO have been reported to upregulate TERT expression (Gessner et al., 2010). AML originates from small populations of leukemia stem cells (LSCs) that have extensive self-renewing capacity but tend to be resistant to chemotherapy and contribute to relapse. It can be predicted that LSCs might be especially

dependent on telomerase activity to sustain long-term replicative capacity and that inhibition of telomerase activity may be a potential approach to target LSC self-renewal. In this issue of Cell Stem Cell, Bruedigam and colleagues (Bruedigam et al., 2014) examined the role of telomerase in AML LSC function and demonstrated an essential role for telomerase in the maintenance of functional LSCs (Figure 1). Using mice depleted of the RNA component of the telomerase (Terc/) and a well-defined genetic model of MLL-AF9-induced AML, they showed that telomerase deficiency greatly compromised self-renewal of LSCs. Although AML developed with full penetrance, suggesting that telomerase is not essential for leukemia initiation, telomerase deficiency greatly reduced leukemia burden and the frequency of functional LSCs measured in limiting dilution and secondary transplantation assays. Similar effects were detected using additional AML models, including the AML1-ETO/KRASG12C AML and BCRABL/NUP98-HOXA9 AML, suggesting that the effects of telomerase deficiency were independent of the oncogenic driver of AML. These results support an

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Cell Stem Cell

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important role for telomerase addition to telomerase-mediSelf-renewal activity in maintaining the ated mechanisms, telomere long-term self-renewing replimaintenance can be carried LSC TERT T Telomerase inhibition cation capacity of LSCs. out through Alternative T TERC Terc/ LSCs demonLengthening of Telomeres DNA damage strated chromosomal insta(ALT) and recombinationbility, which is marked by based mechanisms. These p53 activation increased end-to-end chroalternative mechanisms Telomere length Cell cycle arrest AML mosomal fusions and is could possibly influence the Apoptosis associated with increased sensitivity or confer resisapoptosis and induction of tance to telomerase inhibition the p53 regulatory network. and should be evaluated in Figure 1. Telomerase Maintains Leukemia Stem Cell Long-Term Self-Renewal Gene expression profiling future studies. Finally, the Telomerase inhibition either genetically (via Terc-deletion or shTERT) or indicated that telomerase variability in telomere length pharmacologically (via imetelstat) limits LSC self-renewal capacity through deficiency in LSCs is associand telomerase activity cell cycle arrest, p53 activation, DNA damage, and apoptosis. Inhibition of telomerase represents a promising approach to combat long-lived and cheated with aberrant cell among different subclones of moresistant LSCs. cycling, DNA replication and LSCs in individual AML parepair, and increased apotients may affect their susptosis. Notably, depletion of p53 using Importantly, addition of imetelstat to ceptibility to telomerase inhibition and shRNA partially restored colony formation chemotherapy reduced or delayed leuke- could be an important determinant of and rescued apoptosis, markedly ac- mia relapse compared to chemotherapy effectiveness in eliminating resistant LSC celerated development of AML, and alone. These results suggest that telome- populations. completely rescued LSC serial-engraft- rase inhibition may be useful to prolong Cancer stem cells constitute the roots ment capacity. These results indicate remission and reduce relapse in AML. of a board spectrum of malignancies that telomerase loss induces chromo- These results are timely because imetel- and their resistance to existing anticancer somal instability and p53 activation that stat is currently being evaluated in clinical treatment represents a significant barrier results in loss of LSC function and leuke- trials for a number of malignancies, to successful disease eradication and mia progression. including myelofibrosis, and trials in AML cure. Attacking the telomere protection Importantly, the relevance of these find- are planned for the future. mechanism may represent an effective The finding that LSCs rely on telome- approach to a variety of malignant stem ings to human AML was also evaluated. This demonstration is critical because of rase to maintain their extensive prolifera- cells. Caution must be exercised to well-recognized differences in telomere tive and self-renewal potential raises consider potential long-term effects on dynamics in mice and men. The telomeres important questions as to what defines various normal tissue stem cells. Although of most mice are 5 to 10 times longer the sensitivity and responses to telome- the current study did not reveal toxicities those of humans, despite having a life rase inhibition. It is noteworthy that of imetelstat toward normal tissues, span that is 30 times shorter, and this among the patients studied, the highest hepatotoxicity was identified as a potendiscrepancy can result in significant sensitivity to imetelstat seemed to corre- tial concern in ongoing clinical trials. interspecies differences in response to late with an enrichment of telomerase- Nevertheless, the study by Bruedigam telomerase deficiency. Profiling human deficient gene expression signature. It et al. elucidates the importance of telomeAML patient samples revealed that a will be important to validate this signature rase in maintaining LSC self-renewal and gene signature associated with telome- as a potential predictor of response in a replication potential and supports the porase-deficient LSCs predicted favorable larger group of patients and in future clin- tential application of telomerase-targeting outcome. Functional studies using ical trials. TERT is overexpressed in sub- therapy to eliminate LSCs in AML and TERT-shRNA in the MM6 human AML sets of AML such as t(8;21), inv(16), and possibly target other types of cancer cell line confirmed that telomerase knock- MLL-rearranged AML, and in the future it stem cells. down inhibited LSC function in vitro and in will be important to determine the relaxenograft transplantation experiments. tionship between TERT expression and Finally, imetelstat (GRN163L), a pharma- sensitivity to telomerase inhibition. The REFERENCES cological telomerase inhibitor, showed authors demonstrated that p53 contribBruedigam, C., Bagger, F.O., Heidel, F.H., Kuhn, significant activity against primary human utes to the inhibition of LSC function but C.P., Guignes, S., Song, A., Austin, R., Vu, T., AML cells. Imetelstat is a lipid-conjugated additional p53-independent mechanisms Lee, E., Riyat, S., et al. (2014). Cell Stem Cell 15, 13-mer oligonucleotide sequence that is are also involved. Telomerase has been this issue, 775–790. complementary to and binds to the RNA implicated in additional noncanonical Gessner, A., Thomas, M., Castro, P.G., Bu¨chler, L., template of telomerase (Herbert et al., functions such as transcriptional regula- Scholz, A., Bru¨mmendorf, T.H., Soria, N.M., Vormoor, J., Greil, J., and Heidenreich, O. (2010). 2005). Administration of imetelstat pre- tion (Ye et al., 2014) and ribosomal DNA Leukemia 24, 1751–1759. vented AML development in primary transcription (Gonzalez et al., 2014). It human xenografts and leukemia progres- will be interesting to investigate whether Gonzalez, O.G., Assfalg, R., Koch, S., Schelling, A., Meena, J.K., Kraus, J., Lechel, A., Katz, S.F., sion remained significantly delayed for these functions of telomerase contribute Benes, V., Scharffetter-Kochanek, K., et al. weeks after suspension of treatment. to the effects of telomerase inhibition. In (2014). Nat. 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Cell Stem Cell

Previews Herbert, B.S., Gellert, G.C., Hochreiter, A., Pongracz, K., Wright, W.E., Zielinska, D., Chin, A.C., Harley, C.B., Shay, J.W., and Gryaznov, S.M. (2005). Oncogene 24, 5262–5268. Pucci, F., Gardano, L., and Harrington, L. (2013). Cell Stem Cell 12, 479–486.

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The Nucleolus Gets the Silent Treatment Andrew P. Feinberg1,* 1Center for Epigenetics and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2014.11.017

 et al. (2014) and Kim et al. (2014) provide insight into how global heteroIn this issue of Cell Stem Cell, Savic chromatin condensation and LIN28 sequestration in embryonic stem cells are independent mechanisms regulating pluripotency in the oft-overlooked nucleolus. Even though we have been aware of the nucleolus for 180 years, and it is the largest structure in the nucleus, it gets little attention compared to the nucleus (15,000 versus 374,000 cites). And much less attention is paid to its role in epigenetics (25 Pubmed citations) or heterochromatin (800 citations) compared to its role in ribosomal gene transcription. That’s actually a bit strange for both historical and biological reasons. Historically, because in the 1930s, Emil Heitz described heterochromatic satellite regions near the nucleolus, and Barbara McClintock described the nucleolar-organizing region’s association with heterochromatin. And biologically, because most ribosomal genes are actually not expressed in normal cells, and their silencing by heterochromatin is necessary to prevent chromosomal rearrangements and excessive growth. In this issue of Cell Stem Cell, two papers now provide insight into independent nucleolar mechanisms regulating pluripotency in embryonic stem cells (ESCs). Much as people now widely accept the importance of the nuclear membrane in organizing heterochromatin structure, and emerging work connects 3D genome organization with regulation of ESC fate (reviewed in Gorkin et al., 2014), the nucleolus has so far gotten the silent treatment in this area. The  et al. addresses one mechwork by Savic

anism for heterochromatin formation during lineage commitment as mouse ESCs  et al., 2014). (mESCs) differentiate (Savic They specifically addressed the role of the long noncoding RNA pRNA, which is the mature form of the processed 2 kb long intergenic spacer (IGS)-rRNA, already known to interact with nucleolar repressor factor TIP5 in generating rDNA heterochromatin. They first showed that ribosomal DNA methylation is decreased in induced pluripotent stem cells and heterochromatin marks H3K9me2/3 and H3K27me3 increase during differentiation; and the changes in both directions are associated with expected increases and decreases in transcription, respectively. They then transfected mESCs with pRNA, which led to TIP5 accumulation in nucleoli and induced heterochromatic rDNA with an increase in CpG methylation and H3K9me2 at the rDNA promoter— and mutagenesis experiments showed that this was dependent on an intact 30 -pRNA stem-loop structure, but not the pRNA-TIP5 association itself. Thus pRNA guides TIP5 to rDNA through its hairpin structure and establishes ribosomal gene heterochromatin. Moreover pRNA expression and TIP5 accumulation led to a dramatic general increase in condensed perinucleolar heterochromatin, as well as total levels of H3K9me2 in the nucleus, an indicator of large-scale heterochromatin formation.

Another paper in this same issue, by Kim et al., identifies a nucleolar targeting mechanism by which a histone methyltransferase—normally these are involved in the epigenetic machinery—affects pluripotency in human ESCs (Kim et al., 2014). The study addresses the mechanism by which the key pluripotency factor LIN28 represses processing of let-7 miRNA (from the pri-let-7 to the pre-let-7) to maintain self-renewal. In the nucleus, LIN28 processing is less well understood than in the cytoplasm, which involves recruitment of TUT4/7 to induce oligo-uridylation of pre-let-7, which appears to both prevent its Dicerinduced processing into mature let-7 and render it susceptible to digestion by the Dis3L2 exonuclease (reviewed in Shyh-Chang and Daley, 2013). The authors were pursuing a relatively recent thread of studies on the nature and effect of methylation of core pluripotency factors. Specifically they investigated the effect of SET7/9 monomethylase on LIN28A, but not the paralogous LIN28B, and identified the methylated residue as lysine 135. Remarkably, the K135 methylation leads to nucleolar localization of LIN28A and sequestration, preventing LIN28A processing independently of TUTase. Interestingly, the K135 site of LIN28A is homologous to the nucleolar localizing signal region of LIN28B, which was already known to

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Pushing the limits: defeating leukemia stem cells by depleting telomerase.

Leukemia stem cells (LSCs), featuring unlimited self-renewal capacity and chemoresistance, are critical cellular targets for new treatments to improve...
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