Targeting telomerase and telomeres to enhance ionizing radiation effects in in vitro and in vivo cancer models

Authors: F. Berardinelli, E. Coluzzi, A. Sgura, A. Antoccia PII: S1383-5742(16)30115-6
DOI: http://dx.doi.org/doi:10.1016/j.mrrev.2017.02.004
Reference: MUTREV 8202

To appear in: Mutation Research

Received date: 18-10-2016
Revised date: 13-2-2017
Accepted date: 14-2-2017

Please cite this article as: F.Berardinelli, E.Coluzzi, A.Sgura, A.Antoccia, Targeting telomerase and telomeres to enhance ionizing radiation effects in in vitro and in vivo cancer models, Mutation Research-Reviews in Mutation Research http://dx.doi.org/10.1016/j.mrrev.2017.02.004

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Targeting telomerase and telomeres to enhance ionizing radiation effects in in vitro and in

vivo cancer models

Berardinelli F.*°#, Coluzzi E.*, Sgura A.*°, Antoccia A.*°

*Dipartimento di Scienze, Università Roma Tre, Rome Italy

°Istituto Nazionale di Fisica Nucleare, INFN, Sezione di Roma Tre, Rome, Italy

#Corresponding author: Francesco Berardinelli, Dipartimento di Scienze, Università Roma Tre, V.le

Marconi 446, 00143, Rome, Italy

email: [email protected]

ABSTRACT

One of the hallmarks of cancer consists in the ability of tumor cells to divide indefinitely, and to maintain stable telomere lengths throughout the activation of specific telomere maintenance mechanisms (TMM). Therefore in the last fifteen years, researchers proposed to target telomerase or telomeric structure in order to block limitless replicative potential of cancer cells providing a fascinating strategy for a broad-spectrum cancer therapy.
In the present review, we report in vitro and in vivo evidence regarding the use of chemical agents targeting both telomerase or telomere structure and showing promising antitumor effects when used in combination with ionizing radiation (IR). RNA interference, antisense oligonucleotides (e.g.,
GRN163L), non-nucleoside inhibitors (e.g., BIBR1532) and nucleoside analogs (e.g., AZT)

represent some of the most potent strategies to inhibit telomerase activity used in combination with IR. Furthermore, radiosensitizing effects were demonstrated also for agents acting directly on the
telomeric structure such as G4-ligands (e.g., RHPS4 and Telomestatin) or telomeric-oligos (T-

oligos). To date, some of these compounds are under clinical evaluation (e.g., GRN163L and

KML001).

Advantages of Telomere/Telomerase Targeting Compounds (T/TTCs) coupled with radiotherapy may be relevant in the treatment of radioresistant tumors and in the development of new optimized treatment plans with reduced dose adsorbed by patients and consequent attenuation of short- end long-term side effects. Pros and cons of possible future applications in cancer therapy based on the combination of T/TCCs and radiation treatment are discussed.

Abbreviations list:

- TMM: Telomere Maintenance Mechanism

- T/TTC: Telomere/Telomerase Targeting Compound

- G4: G-quadruplex

- DSB: Double Strand Break

- ALT: Alternative Lengthening of Telomeres

- GBM: Glioblastoma Multiforme

- AS-ODN: Antisense Oligonucleotide

- TIF: Telomere-Induced Dysfunctional Foci

- RT: Radiotherapy

- DDR: DNA Damage Repair

- IR: Ionising Radiation

- NHEJ: Non Homologous End Joining

- HR: Homologous Recombination

Keywords: Telomeres, Telomerase, G-quadruplex, ionizing radiation, radiotherapy

1.Introduction

Telomeres are nucleoprotein structures organized into heterochromatin domains and located at the end of linear chromosomes, which primary role is to maintain chromosome integrity and thus genome stability [1]. In humans, telomeric DNA consists of conserved, non-coding regions composed by tandem repeats of the G-rich exanucleotide (TTAGGG)n, which are typically 10-15 Kb in length [2] (Figure 1).
In physiological conditions, G-rich sequences (and in particular telomeric DNA) are capable to assume non-canonical DNA helical structures known as G-quadruplexes (G4s) [3]. These structures

are composed by stack of G-quartets, which are planar assembly of four Hoogsten-bonded guanine bases. Great conformational diversity characterizes G4s structure that can be formed by the same (intramolecular) or different (intermolecular) DNA chains and may display different (i) number of stacked G-quartets (two or more), (ii) orientation of G-strands (parallel or antiparallel), (iii) glycosidic conformations of the guanines (syn and anti, depending on the orientation of the N- glycosidic bond), and (iv) loop types that connect the G-strands (lateral, diagonal and propeller). Stacks of G-quartets are stabilized by monovalent cations such as K+ and Na+ and under physiological conditions are very stable and resistant to thermal denaturation. The signature motif predicting G4s formation is characterized by four strands of at least three guanines separated by other bases (G≥3NXG≥3NXG≥3NXG≥3, where N is any nitrogen base). Bioinformatics analysis on human genome revealed that G4 sequence motifs are present in gene promoters, gene bodies, and in repetitive DNA, such as telomeric DNA [4]. Recent evidence support the idea that G4s play a regulatory role functioning in diverse cellular pathways such as DNA replication, gene expression, and telomere protection [5]. However, in higher eukaryotes, telomere protection is mainly achieved through the formation of the telomere loop (t-loop) that involves strand invasion of the terminal single stranded telomeric sequence (G-tail) into internal telomeric repeats of the same chromosome end [6].
Stability of t-loop is guaranteed by the interaction with a number of specific proteins, which form the so-called “Shelterin complex”. This complex is essentially composed by six proteins, TRF1 and TRF2, RAP1, TIN2, TPP1 and POT1 [7] (Figure 1) and plays a crucial role in the t-loop formation and hence in telomere protection [8]. Indeed, Shelterin proteins are abundant at telomeres displaying a remarkable specificity for the telomeric sequences TTAGGG: in particular, TRF1 and TRF2 directly bind the duplex telomere repeats as homodimer whereas POT1 binds the single- stranded DNA at the very end of chromosomes [1].
Telomeres play essentially two major functions in eukaryotes; (i) to protect the end of linear chromosomes avoiding that DNA repair mechanisms may recognize natural ends as DNA damage

(i.e DNA double-strand breaks, DSBs); and (ii) to define the number of cell cycles that a cell may undergo during its life. Indeed, at each round of somatic cell division telomeres get shorter due to the combination of two different factors that are (i) the end-replication problem and (ii) the processing that occur to form the G-tail, generated during the synthesis of the lagging strand [9]. As a consequence, in somatic cells, in which telomere maintenance mechanisms are turned off, about 100-200 bases of telomeric DNA are lost at each replication round [10-12].
Eroded telomeres may be very harmful for the genomic integrity of the cell and therefore cells carrying critically short telomeres activate DNA damage response (DDR) that in turn lead to the block of cell proliferation and eventually to apoptosis [13] (Figure 1). These protective mechanisms avoid the proliferation of potentially precancerous cells, playing a central role in the regulation of cellular lifespan and avoiding malignant transformation [14]. Considering the important roles of telomeres in cellular functions, it is not surprising that multiple (at least two) telomere maintenance mechanisms (TMMs) exist in mammalian cells.
These mechanisms play important roles in physiological functions but may be improperly re- activated during cellular transformation, conferring unlimited replication potential to cancer cells. The most frequent TMM activated in cancer cells (85-90% of the tumors) is the telomerase. Telomerase is a ribonucleoprotein complex that regulates telomere-length maintenance by adding
telomere repeats to the chromosome 3’-end using an RNA template (see section 4 for further details). The remaining 10-15% of the tested tumors display a different TMM called Alternative Lengthening of Telomeres (ALT) based on telomeric recombination [15,16]. The theory of carcinogenesis suggests that an unlimited cell proliferation is necessary for the development of malignant disease, and cancer cells must reach immortality for progression to malignant states. Therefore, the re-activation/activation of TMMs is an essential step for cell transformation.

2.Telomere homeostasis and sensitivity to ionizing radiation

2.1Human radiosensitive syndromes and telomeres

The relationship between sensitivity to ionizing radiation (IR) and telomere function was mainly based on the observations that rare human recessive genetic syndromes, known for their spontaneous chromosomal instability and clinical radiosensitivity, shared defective telomere maintenance. In fact, patients affected by Ataxia-telangiectasia (AT), Nimegen Breakage Sindrome (NBS), Ataxia-telangiectasia-like disorder (ATLD) and Fanconi Anemia (FA) group D, in spite of the different clinical signs, all show peripheral blood lymphocytes carrying critically short telomeres [17-22]. Studies performed on a panel of 11 human fibroblast cell lines and a mouse embryonic stem cell line with different genetic deficiencies (e.g., defective ligase IV, Artemis and Brca1) showed an accelerated rate of telomere shortening compared to cell lines established from normal healthy counterparts, corroborating the link between increased sensitivity to IR and telomere dysfunctions [23]. Furthermore, the cytogenetic analysis indicated that telomere shortening prompted genomic instability characterized by end-to-end chromosome fusions and chromosome bridges, thus suggesting the loss of telomere capping functions [24]. Evidence gained in mouse models deficient in the DDR (see below), reinforced these early studies, thus indicating that telomere maintenance reflects the activity of a subset of genes whose products are strictly connected to the DDR, that is sensing, signalling and repairing of the DNA [25]. Therefore, it was not surprising that several proteins identified originally as part of the DDR were successively found physically associated with functional telomeres. In particular, proteins such as those of the MRN complex (MRE11, NBN, and RAD50), DNA-PKcs, Ku70/80, ATM and ATR are sequestered on telomeric chromatin by TRF2, some of them apparently in a cell cycle dependent manner [29,30]
while others as PARP1, BRCA1, RAD9/Hus1/RAD1, RAD51D etc., have been reported to interact with telomeres [27,31] (Table 1). This led Slijepcevic to propose the so-called “integrative” model in which telomere maintenance plays an integral part in the DNA damage response [26-28].

2.2Telomeres shortening/deprotection and response to IR

Although, studies on telomerase-deficient mice and human cell lines have demonstrated that

telomere shortening enhances sensitivity to chemical genotoxic agents, the molecular basis for this

observation remains yet unclear [32-34]. The generation of TERC-/- deficient mice (mTERC-/-)

allowed for the first time to test the relationship between progressive telomere shortening, telomerase activity, and sensitivity to IR [32,33]. Interestingly, only late generation mTERC-/- mice showed accelerated mortality upon exposure to -rays, increased rate of apoptosis, and cytogenetic damage with respect to the TERC+/+ counterpart [32,33]. The fact that such an effect was not observed in early generation mTERC-/- mice suggested that telomere length, rather than telomerase deficiency, was the primary determinant of radiosensitivity [32,33,35-38]. Only few studies have suggested that telomerase expression per se influences radiosensitivity [39,40], whereas the majority of works gained also in human cells [37,41-43] showed that after the ectopic telomerase expression or telomerase inhibition did not influence the sensitivity to IR, until a measurable change in telomere length was achieved. More recently, Drissi and coworkers demonstrated a greater sensitivity to IR in late-passage primary human foreskin fibroblasts (HFF) carrying short telomeres than in early-passage cells with longer telomeres [31,44].
The increased radiosensitivity as a function of telomere length was also investigated in murine cell lines, in normal lymphocytes, in lymphoblasts and in cells drawn from cancer patients. A consistent reduction in telomere length was observed in the radiosensitive murine lymphoma cells L5178Y-S compared with the radioresistant parental cells L5178Y, possessing 7 kb and 48 kb, respectively [45]. However, such a relationship was not confirmed in the same cell lines belonging to different stocks [46]. Concerning peripheral blood lymphocytes, it has been shown that telomere length modulates chromosome radiosensitivity in vitro in healthy individuals, as the patient cohort with short telomeres presented higher frequencies of IR-induced micronuclei (MN) when compared to the long-telomeres cohort [47]. Again, an inverse correlation between mean telomere length and in vitro radio-induced cytogenetic damage was reported in lymphocytes drawn from breast cancer patients [48]. Ectopic expression of the catalytic subunit of TERT in human fibroblasts with different telomere lengths showed that only cells with short telomeres acquired resistance to

genotoxic agents including IR [42]. Beside human fibroblasts, a relationship between telomere size and increased radiosensitivity was also reported in non-transformed human epithelial cells in which telomere-DSB interaction occurred prevalently in chromosomes with the shortest telomeres [49]. On the other hand, enforced telomere elongation beyond a certain length significantly decreased cell clonogenic potential of human tumor cell lines. Such susceptibility to IR was dosage-dependent and increased at telomere lengths exceeding 17 kb [50]. Furthermore, it was reported a significant negative correlation between telomere length and radiosensitivity in 15 human carcinoma cell lines from various tissues [51].
Of note, telomere loss in individual cells is a more prognostic marker of in vitro radiosensitivity rather than the mean telomere length [48], in agreement with the notion that the persistence of very short telomeres, rather than the average telomere length per se, has a deep impact on a number of cellular functions such as senescence, cell cycle regulation, chromosomal instability, and even ALT activation [36,48,15,52,53].
The biological mechanism by which telomere shortening (or telomere loss) radiosensitizes cells are still not completely understood and therefore different hypotheses have been proposed. In this respect, no impairment of the DSBs repair pathways (i.e., non homologous end-joining (NHEJ) [32,33] and homologous recombination (HR) [32]) were detected in short telomeres, telomerase- deficient (mTERC-/-) mice, thought the radiosensitivity of telomere dysfunctional Mouse Embryonic Fibroblasts (MEFs) was accompanied by delayed DSBs repair kinetics, persistent chromosomal breaks, and cytogenetic profiles characterized by complex chromosomal aberrations and massive fragmentation [32]. Successively, combined telomeric Fluorescence In Situ Hybridization (FISH) and multicolor FISH (mFISH) approaches were applied to perform a detailed cytogenetic analysis in irradiated mTERC-/- MEFs [54]. The authors found that chromosomes with shortest telomeres were more frequently involved in chromosome aberrations, and furthermore such aberrations were not restricted to end-to-end fusions but also comprised the interaction between eroded telomeres and genomic radiation-induced chromosome breaks. This suggested that the

synergy between telomere shortening and IR was mediated by the capability of short dysfunctional telomeres to interfere with the properness of radiation-induced DSBs repair, thus giving rise to additional chromosomal aberrations originating from telomeres-DSB rearrangements [54].
A further hypothesis was based on the observation that late-passage HFF with short telomeres had attenuated peak levels of H2AX phosphorylation and that the kinetics of H2AX phosphorylation and dephosphorylation were altered after IR-exposure. The authors proposed that telomere shortening is associated with chromatin structural changes (i.e., histone acetylation and methylation profiles suggestive of compacted chromatin) that limit the access of activated ATM to its chromatin-bound downstream targets, such as H2AX, SMC1, and NBN, with a consequent effect on cell survival [44].
As reported in the above sections, several are the telomere-localizing proteins that contribute to the formation and stabilization of the t-loop structure and consequently to the telomere functionality. Therefore telomere dysfunction can arise as a result of the natural/induced telomere shortening, of DDR proteins loss of function (Table 1), or of Shelterin complex proteins loss of function (Figure 1). For some of them, a relationship with response to IR has been provided.
Increasing evidence indicates that POT1 plays a role in telomere overhang protection, DNA damage signalling at telomeres and regulation of cell cycle progression [55,56]. Interestingly the mRNA level of two splicing variants of POT1, and namely the variant1 (POT1 v1) and the variant 5 (POT1 v5), were associated with telomere length and radioresistance in colon and gastric adenocarcinoma cell lines [57]. Authors stated that reduced POT1 expression could be associated with telomere dysfunction and may be a marker of individual radiosensitivity [57]. Indeed, the over-expression of POT1 was linked to radioresistance and increased telomere length in cell derived from the human laryngeal cancer Hep-2 and POT1 silencing reverted the cellular phenotype [58].
Li and coworkers found that the treatment of normal human T lymphocytes and fibroblasts with γ- rays led to significant shortening of telomeres, down-regulation of telomerase activity, and

diminished expression of telomerase reverse transcriptase and the telomere binding proteins TPP1 and POT1 [59].
TRF1 gene targeting disruption in the chicken DT40 cells led to a sustained sensitivity to IR in terms of cell killing and delayed rejoining of DSBs compared to wild-type counterparts [60]. In addition, it has been recently reported that TRF1 and TPP1 were upregulated at both gene and protein level in radioresistant breast cancer cells [61]. Regarding TPP1, it has been observed that its expression was higher in radioresistant Hep-2R cells than in the more sensitive Hep-2 cell line. In addition, a link between overexpression of TPP1, radioresistance and telomere lengthening was reported in telomerase-positive HCT116 cells [62,63].
Overall, data here reported underlined the tight link between the maintenance of telomere length/structural integrity and the sensitivity to IR, thus opening the way to the use of chemical strategies interfering with enzymes/mechanisms involved in telomere homeostasis, and consequently, to the sensitization of tumor cells to the radiation treatment.

3.T/TTCs to improve radiation effects

Radiotherapy (RT) is one of the safest and most effective techniques used to fight cancer and, to date, about 50% of all patients suffering from solid tumors receive IR. However, the therapeutic efficacy is tightly dependent on the sensitivity of the tumor that receives radiation exposure. Different type of cancers display high resistance to IR and for some of them (e.g. glioblastoma multiforme, GBM) radioresistance is one of the main reason of their poor prognosis. In this perspective, promoting tumor cell sensitivity to IR could significantly enhance the treatment outcome and quality of life for patients. Unfortunately, the mechanisms responsible for tumors radioresistance remain elusive and several studies have shown that radioresistance involves different cellular processes such as DNA repair, upregulation of pro-survival proteins, change in cellular metabolism, hypoxia and influence of tumor microenvironment [64].
As discussed above, studies performed in the last fifteen years revealed that telomeres are

implicated in the maintenance of genomic stability, repairing of damaged DNA, and, most importantly play a pivotal role in the regulation of cellular radiosensitivity/radioresistance. Therefore, the application of telomere/telomerase targeting compounds (T/TTCs) in combination with RT may represent a valuable approach to enhance the effect of IR, increasing the efficacy of RT and reducing radiation dose delivered to patients, thus minimizing side effects to normal tissue. Targeting telomeres through the use of telomerase inhibitors or through compounds able to destabilize telomeric structure imply activation of different cellular responses that, ultimately, converge to telomere function impairment and thus increased radiation response (Figure 2).

4.Targeting telomerase to enhances radiosensitivity

The main activity of the telomerase is to maintain telomere length, and therefore telomerase was the most obvious target to be spoiled in order to block proliferation of cancer cells. An increase in telomerase activity has been often directly correlated with uncontrolled growth of cells, which is a known hallmark of cancer [65], and in many reports a direct correlation between telomerase activity and cellular radioresistance has been demonstrated. Based on the experimental evidence that links telomere homeostasis to radiation sensitivity, pharmacological targeting of telomerase represents a promising tool for cancer therapy, not only per se but also in combination with IR (table 2). Telomerase holoenzyme, formed by the catalytic subunit (TERT) and the RNA template subunit (TERC), is regulated at numerous levels such as epigenetic regulation, transcriptional and post- translational processing, intracellular compartmentalization, recruitment and accessibility to its substrate. In turn these regulation mechanisms provide different opportunities for therapeutic targeting [66], among them RNA interference (RNAi) of the telomerase subunits, antisense oligonucleotides targeting the RNA template, catalytic inhibitors and nucleoside analogs were successfully tested in combination with IR (Table 1 and Figure 3). For completeness, it should be mentioned that efficient telomerase-based therapies may generate selective pressure favoring adaptive resistance mechanisms. For instance, ALT and telomerase are non mutually exclusive

mechanisms and can coexist to maintain telomere length in mammalian cells [67,68]. Indeed, it is not unlikely that telomerase-targeting therapeutics may favor the emergence of ALT-dependent cancer cell survival and proliferation, posing new therapeutic challenges (see section 6).

4.1RNA interference

Post-transcriptional gene silencing by RNAi leads to reduced mRNA and thus protein of a target gene, and this approach has been used to target telomerase. Various siRNAs have different efficiency in suppressing telomerase expression, and this method has been largely employed to inhibit telomerase activity either by targeting TERT or TERC.
Nakamura and coworkers demonstrated that introducing a retroviral vector encoding the TERT-specific short hairpin (shRNA) in HeLa and SiHa human uterine cervical cancer cell lines, a stable suppression of hTERT expression and a decrease in telomerase activity was obtained. Cells silenced for hTERT showed a progressive reduction in telomere length, a slower proliferation rate and underwent senescence after considerable population doublings [40]. In addition, authors showed that reduced expression of TERT silencing potentiated the effect of IR and chemotherapeutics able to induce DSBs. Furthermore, the combined approach using TERT shRNA and DSBs-inducing agents such as IR, Etoposide, Bleomycin and Doxorubicin resulted in a synergistic reduction of cell survival and growth rate in cervical cancer cell lines, whereas the combination with Paclitaxel and Cisplatin did not. The same results were obtained in human colon cancer cell HCT116 displaying TERT haploinsufficiency and telomere dysfunction [69]. Although some reports suggested the interference of dysfunctional foci with DSBs repair as the mechanism responsible [33], others proposed a more direct role of telomerase in enhancing the DDR through the increase of the dNTP pool and the induction of genes such as Rad51, MLH1 and MSH6 [70] or by the telomerase mediated recruiting of DNA repair proteins to the site of the DNA lesion [71]. The synergistic interaction between telomerase depletion and IR was confirmed by other studies in cervical carcinoma cancer cells [72-74]. SiHa and HeLa cells silenced for TERT or TERC displayed

a rapid inhibition of cell proliferation and an increased radiosensitivity, as evaluated by surviving fraction experiments [73,74]. Linear quadratic model calculation from surviving fraction experiments showed an increase in the  parameter from 0.26 in controls SiHa cells to 0.45 in TERT silenced cells, whereas the  parameter moved from 0.02 to 0.03 [74]. For Hela cells silenced for TERC author reported a radiation enhancement ratio of 2.4 and a significantly reduced tumorigenic potential of cells exposed to combined treatment [73].
However, telomerase is active not only in tumor cells, but also in germ, nerve stem and haemopoietic stem cells [75,76]. Therefore, as discussed later, a possible Achille’s heel in the targeting of TERT is the risk to affect normal telomerase positive cells that should be hopefully spared from treatment. In this respect, Wang and co-authors in 2007 [72] developed a strategy to downregulate TERT expression in a specific manner by constructing survivin promoter-driven TERT siRNA eukaryotic expression vectors. Survivin is a member of the inhibitor of apoptosis (IAP) protein family expressed in most of human cancers but undetectable in normal differentiated cells [77,78]. The authors observed a decreased telomerase activity due to the inhibition of hTERT at both mRNA and protein levels and an increased caspase-3 activity and apoptosis [72]. Results were strengthened by in vivo studies in which HeLa cells, transfected with the survivin promoter- driven TERT siRNA, were injected in athymic BALB/c nude mice. One month after the injection most of the mice did not develop any tumor and only 17% of them developed tumors three times smaller than those in control mice [72]. Very interestingly, authors reported that telomerase suppression in HeLa cells determined a significant increase in radiosensitivity as assessed by colony forming assay at doses of 2, 4, 6 and 8 Gy of X-rays and indicated the surviving promoter-driven TERT downregulation as a promising tool for the radiosensitization of human cervical carcinoma [72]. In support of this notion, it has been shown that ectopic overexpression of TERT gene confers karyotypic stability in irradiated human fibroblasts [79].

4.2Antisense oligonucleotides

Another strategy that was developed to achieve inhibition of telomerase activity relied on the use of antisense oligonucleotides (AS-ODNs), which are short single-stranded DNA (ssDNA) able to block mRNA translation thanks to their sequence complementary to the sense RNA. Although, this approach could be used to target either TERT or TERC subunits, oligomers targeting TERC resulted more effective than those targeting TERT. One of the possible explanations is that the template region of TERC possesses a high accessibility to hybridization with AS-ODN since this region is physiologically exposed to favor the telomeric G-tail binding. Moreover, hTERT mRNA is rich of GC residues (66%) that may render hybridization less proficient [80]. The results obtained after TERC AS-ODNs treatment in PC3 prostate cancer and in MCF-7 breast cancer cells indicated a reduction in telomerase activity accompanied by telomere shortening and decreased cell proliferation [80]. In 2006 Ji and coworkers used an AS-ODN targeting nucleotides in position 38- 50 of TERC subunit [81] and evaluated the effects in combination with IR on U251 glioma cells [82]. AS-ODN caused a stable suppression of hTERC and the consequent inhibition of telomerase activity. In addition the combined treatment with -rays led to an increase in DNA damage and a reduction in surviving fraction with D0 values (D0 is the dose necessary to reduce surviving fraction to 37%) of 2.77, 2.65 and 2.02 Gy in controls cells, cells exposed to non specific and TERC specific oligos, respectively [82].
Among AS-ODN, the most promising molecule is the GRN163L oligo (entered in clinical trial as Imetelstat). GRN163L is a lipid-based conjugate of the first-generation oliogonucleotide GRN163 [83], and consist of a 13-mer N3’P5’ thio-phosphoramidate oligonucleotide that is covalently attached to a C16 (palmitoyl) lipid moiety [84]. GRN163L inhibits the biochemical activity of telomerase through the binding to the telomeric template of hTERC, blocking telomere access to telomerase and thereby acting like a conventional pharmaceutical drug. To date, GNR163L appears to be the most successful AS-ODN developed because of its stability, resistance to nuclease degradation, high specificity for DNA and RNA (but not for proteins), and high bioavailability and cellular uptake [85,86]. Telomeres inhibition by GRN163L has been shown in many human tumor

cells such as lung [87,88], breast [85,86,89], prostate [83], liver [90], brain [91], bladder [92], and hematological malignancies including multiple myeloma and lymphoma [93,94], in both cell culture systems and mouse xenograft models. Interestingly, no alterations in telomere length and cell proliferation were detected in normal telomerase-negative HMEC and CFB8 human endothelial cells, thus suggesting an effect almost restricted to tumor cell lines [85]. Moreover, GRN163L treatment followed by chemotherapeutics or IR evidenced the beneficial effects of combined strategies. In particular, experiments on breast carcinoma cells indicated that GRN163L efficiently reduced telomerase activity, telomere length and cell proliferation as single agent. While telomerase expression was completely inhibited in few days, effects on telomere length, and proliferation became evident only after 20-42 days from the first drug administration. Interestingly, only long- term GRN163L treatments synergistically enhance the effect of X-rays, indicating that telomere erosion, and very likely telomere dysfunction, is an essential step to get the radiosensitization [86]. These results were confirmed in vivo in a xenograft mouse model. Data showed that mice injected with GRN163L-treated cells and then exposed to 6 Gy of X-rays resulted in a highly statistically significant difference in tumor growth compared to mice receiving either IR alone or IR combined with the control oligo. After 80 days from injection, all mice (100%) belonging to the GRN163L + IR group survived compared to the other groups that displayed a survival comprised between 14 and 52% due to excess tumor burden [86]. Recently, Ferrandon and colleagues confirmed the efficacy of GRN163L as radiosensitizer in a murine orthotopic model of human GBM (U87MG cells). The potent telomerase inhibitor administered through the intra-peritoneal route was able to reduce telomerase activity in the core of the tumor, to reduce tumor volume and to increase the response to radiation therapy in terms of tumor volume regression as evaluated by magnetic resonance imaging and survival increase [95]. Although, authors proposed the clinical evaluation of the inhibitor in combination with RT, to date GRN163L has been tested only as single agent or in combination with other chemotherapeutics in 16 clinical trials (i.e., 8 phase I, 7 phase II and 1 phase

3) on diverse types of tumors including myeloma, tumors of the breast, lung, brain and central nervous system (www.clinicaltrial.gov).

4.3Non-nucleoside inhibitors

A variety of non-nucleoside drugs are also able to inhibit telomerase such as Epicatechin derivatives Epigallocatechin gallate (EGCg) and MST312 both able to strongly and directly inhibit telomerase [96-98]. EGCg has been identified as having many biological functions beside telomerase inhibition, including the block of cancer cell growth [99], cell cycle arrest [99-101], pro-apoptotic activities [99,102,103], inhibition of invasion and metastasis [104,105], and anti-angiogenic properties [103,106]. Moreover, several reports have proposed that EGCg can sensitize cancer cells to IR [107], by potentiating the effects of RT in breast cancer patients [108], sensitizing endothelial brain cells to IR [109], increasing the radiation-induced apoptosis and reducing cell growth in IM-9 (multiple Myeloma) and K-562 (chronic myelogenous leukaemia) cells [110]. In 2002, Seimiya and collegues [97] described three new analogs of EGCg, namely MST-312, MST-295 and MST-199, showing a greater inhibitory effect on telomerase and inducing cell growth reduction, telomere shortening and senescence phenotype in U937 monoblastoid leukemia cells [97]. Very recently MST-312 has been tested in combination with X-rays in hepatoma HepG2 cells with encouraging results [106]. Data indicated that the combined treatments was able to reduce HepG2 survival and to delay -H2AX dephosphorylation kinetic through the impairment of HR repair, as shown by the failure of RAD51 nuclear translocation [111]. The accumulation of unrepaired DNA damage, in addition, increased p53 level and, as a consequence, the mithocondrion-mediated apoptosis [111]. The small-molecule BIBR1532 is one of the most potent selective non-competitive, non-nucleoside pharmacological inhibitor targeting the telomerase core components [112,113]. It has been proposed that BIBR1532 might influence the translocation of the enzyme on the substrate or alternatively the dissociation of the enzyme from the telomeric DNA [113]. The first report regarding the inhibitory activity of BIBR1532 was published in 2001 by Damm and collegues and

showed that nanomolar concentrations of the inhibitor were able to repress telomerase activity and to shorten telomeres in long term cultures (60-200 population doublings, depending from the cell line) of either tumoral p53-proficient (NCI-H460, lung carcinoma) or p53-deficient (HT1080, fibrosarcoma; MDA-MB231, breast carcinoma; DU145, prostate carcinoma) cell lines [112]. Long- term inhibition of telomerase led to senescence and telomere dysfunction with the onset of chromosomal aberrations such as telomere fusions [112]. Although no data are available on the combined effect of BIBR1532 and IR, some reports indicated the efficacy of BIBR1532 and radiomimetic drugs combined treatments in drug-resistant cancer cells. Interestingly, long-term administration of 2.5 M BIBR1532 sensitized HL60/WT and HL60/MX2 (Etoposide resistant) to the radiomimetic drug Etoposide. Longer administration of BIBR1532 led to progressive telomere shortening and reduced cell growth in the presence of Etoposide (IC25 values of 0.175 µM and 4.92 µM for HL60/WT and HL60/MX2, respectively). Same results were obtained in MCF7/wt and MCF7/MlnR (Melphalan resistant) in response to the radiomimetic drug Doxorubicin and the alkylating agent Melphalan [114]. Of note, BIBR1532 is also able to sensitize breast cancer cells to mitotic spindle poisons such as Taxol, which does not induce clastogenic effects [115].

4.4Nucleoside analogs

Tested originally in Tetrahymena, nucleoside analogs, such as 3-azido-2,3-dideoxythymidine (AZT), act as chain-terminating inhibitors of reverse transcriptase and were among the first drugs to be tested for their ability to inhibit telomerase [116,117]. Despite AZT is a no-specific telomerase inhibitor [116], enduring AZT treatment results in inhibition of telomerase activity, progressive telomere shortening, and increased p14(ARF) expression [118]. In 2007, Zhou and coworkers demonstrated radiosensitizing properties of AZT in U251 glioma cells. Authors reported that the combined AZT and X-rays treatment delayed DNA repair kinetics of SSB and DSB in the first 6 hours after irradiation and decreased the surviving fraction (D0 value of 1.8 and 1.4 Gy in X-ray

treated cells and AZT + X-ray treated cells, respectively) [119]. These data were in line with those reported by Hiraoka and collegues in the Chinese hamster V79 cell line [120].
A further very effective nucleoside telomerase inhibitor is the 6-thio-7-deaza-2deoxyguanosine 5- triphosphate (TDG-TP), which is not only more specific than AZT but also shows a lower IC50 value (0.06 M) [121]. However, to our knowledge, no data are available on the effect of combined approach with either IR or radiomimetic compounds. Moreover, despite some interesting and promising results very poor data are present in the recent literature concerning this class of inhibitors, indicating that current interest for these compounds in single as well as combined treatments is relatively limited.

5.Targeting telomeres to improve radiosensitivity

Among the approaches developed to specifically target telomeres, the stabilization of the telomeric G4 structure, the depletion of proteins involved in telomeric capping and the use of T-oligos that mimic unprotected chromosome termini represent the most effective strategies (Figure 3). The strategies developed to induce telomere dysfunction and the experimental evidence of their radiosensitizing effects are discussed in the following paragraphs and the proposed or possible use of such approaches in future therapeutic applications is highlighted (Table 3).

5.1G4 ligands

Data supporting the in vivo G4 formation in oncogene promoters and telomeres emerged from the use of small molecules able to bind and stabilize G4s. These molecules create a physical encumbrance and lead to the replicative fork arrest at the telomeric DNA [122,123]. As a result, G4
stabilization determines telomere dysfunction and telomeric protein (e.g., POT1 and TRF2)

detachment.

G4 targeting molecules have been largely based on polycyclic planar aromatic compounds and to

date several works have reported their efficacy in different cancer cell types. Telomeric G4 ligands

(e.g BRACO-19, Telomestatin and RHPS4) exert anti-tumoral effects when administered in single treatments [124-126] or in combination with genotoxic drugs or chemotherapeutics both in vitro
and in vivo settings [127-131]. For instance, the trisubstituted acridine BRACO-19 displayed synergistic antitumor effect when combined with Paclitaxel in xenografted tumor derived from the human vulval carcinoma cells A431 [127]. Similarly, the BRACO-19 analog AS1410 showed synergistic activity in vitro and significant reduction of tumor growth in vivo, when combined with Cisplatin [130]. Exposure of K562 cells to Telomestatin (SOT-095) enhanced the sensitivity to Imatinib, Doxorubicin, Mitoxantrone and Vincristine [128]. The pentacyclic acridine RHPS4 is a highly potent and specific telomeric G4 ligand, initially proposed as a mere telomerase inhibitor [132]. RHPS4 is very effective as single agent in cancer growth inhibition as shown in vitro and in vivo in both telomerase and ALT positive cells [126,132-137]. Different works showed that RHPS4 exerts a synergistic effect if combined with radiomimetic compounds such as Bleomycin and Camptothecin [129], that interestingly are both able to induce DSBs [138]. More recently, has been shown that the administration of Topo-I inhibitors followed by the treatment with different G4 ligands (i.e., RHPS4, CORON and PIP-PIPER) determines a very powerful effect on colon carcinoma cells in terms of cell survival [139]. Although RHPS4 has been considered a very strong candidate to enter clinical investigation, in 2013 a preclinical study indicated RHPS4-induced cardiac toxicity in guinea pigs [140]. Indeed, the compound showed significant interaction with the adrenergic M1, M2 and M3 muscarinic receptors and also classified as a highly potent inhibitor of the hERG (human Ether-a-go-go Related Gene) tail current as tested in patch clamp experiments [140], which can be predictive of cardiovascular complications in clinical development [141]. Very recently, new RHPS4 derivatives were proposed to minimize off-target effects, to improve toxicological profiles and to maintain telomere G4 specificity [140,142]. As far as we know, no data on the combined effects of RHPS4 derivatives with either chemical or physical DSBs inducing agents are available. An additional fascinating characteristic of RHPS4 is its ability to inhibit tumor cell growth in vitro and in vivo through a mechanism consistent with a cancer stem cell specific

targeting. In 2007, Phatak and colleagues proposed that combined treatments using RHPS4 in combination with a debulking agent such as Taxol have a highly potent effect due to the targeting of both stem cell compartment and the tumoral differentiated population [135]. Very recently, stem cell targeting by RHPS4 was also demonstrated in colon cancer stem cells [143].
Among G4 ligands, only few (i.e., TAC, RHPS4 and Pt-ctpy) have been tested for treatments in combination with IR. TAC consists of N-methylated triflate derivatives of 4,6-bis-(6-(acrid-9-yl)- pyridin-2-yl)-pyrimidine and details for his synthesis were described in [144]. The TAC compound was shown to partially inhibit cell growth in long-term proliferation assay in two different patient- derived GBM cell lines. Moreover, authors reported a significant induction of apoptosis, 53BP1 foci formation and hTERT overexpression at a concentration of 2µM. Interestingly, the pretreatment with TAC 0.5 or 1 µM was able to induce a reduction in cell survival in samples exposed to γ-rays in a dose range of 2-8 Gy. The combined treatments increased the residual DNA damage after 24 hours from irradiation, induced a G2 block and increased chromosomal aberrations such as dicentrics, acentric fragments and chromatid-type exchanges [144], providing the first evidence that exposure to a G4 ligand radiosensitizes human GBM cells in vitro. However, some questions remained unresolved such as the mechanism by which cells became radiosensitive after the treatment with the G4 ligands and the possible involvement of the telomere structure in the chromosomal aberrations observed. Insights about these issues were provided by a work on the radiosesnitizing effects of the RHPS4 [137]. Authors demonstrated that RHPS4 was able to induce Telomere Dysfunctional Foci (TIF) in a panel of GBM cell lines in a time- and concentration- dependent fashion and that the number of TIF linearly correlate with radiosensitivity. Surviving fraction experiments demonstrated a strong radiosensitizing effect of RHPS4 and a synergistic interaction between the G4 ligand and X-rays. The 2 Gy-surviving fraction, which is the dose used daily in the standard care treatment of GBM patients, was 2.5-fold lower in RHPS4-pretreated sample than in RHPS4-free samples. The increased cell death was due to a delay in the radio- induced DNA damage repair kinetic and to the formation of lethal chromosome aberrations

involving telomeres such as telomeric fusions. Interestingly, authors reported a higher resistance to RHPS4 in normal primary fibroblasts (MRC5 and AG01522) when compared to a panel of GBM cell lines, in particular at doses comprised between 0.1 and 0.4 µM (mean IC50 value of 1 and 0.5 µM in fibroblasts and GBM cells, respectively) [137]. A weak correlation between cellular sensitivity to RHPS4 and fraction of very short telomeres was found [137], but rather than telomere length per se RHPS4 sensitivity is very likely determined by different factors (e.g. normal function of telomeric proteins, frequency of short or undetectable telomere signals, and telomerase activity) that in toto determine the telomeric status of the cell. However, the different sensitivity observed may open the possibility of an interesting therapeutic window for the use of RHPS4 or similar compounds. These data demonstrated that the radiosensitizing effect of RHPS4, and in principle of all telomere targeting drugs, effectively reside in the ability to render dysfunctional a subset of telomeres, specifically in tumor cells [137]. However, telomeric destabilization determined by RHPS4 could be only one of the triggers that prompt the radiosensitization observed. Another intriguing explanation may reside in the specific targeting of either bulk differentiated cancer cells or cancer stem cells by IR and RHPS4 administration, respectively.
Very recently the first evidence of the in vivo efficacy of a G4 ligands in combination with IR was provided [145]. Submicromolar concentration of Pt-ctpy, a second-generation G4 ligand belonging to the tolyterpyridine-metal complexes family, was used to treat GBM and non-small cell lung cancer cells (NSCLC). The molecule in single treatments reduced cell growth with an accumulation of cells in the S-phase and a block in G2/M especially in GBM cells. Pretreatment with Pt-ctpy was able to radiosensitize both GBM and NSCLC cells in vitro and the same results were confirmed in in vivo xenograft tumors as shown by significant decrease in tumor growth rate and the increase in mice survival in combined treatment group (Pt-ctpy 2mg/Kg/die for 10 days + 15 Gy X-rays delivered at day 5 in a single dose) when compared to control and single treatments groups (only drug administration or IR) [145].

5.2Telomeric proteins

Another possibility to harm the integrity of telomeres consists in the specific targeting of genes directly or indirectly involved in telomere stability. A great number of DDR genes, senescence and telomere maintenance are potentially good candidates to be target genes for radiosensitization [21]. To date, four out of six genes that are involved in shaping the shelterin complex (TRF1, TRF2, POT1, TPP1) have been associated with cellular or organismal radiation sensitivity and among them TRF2 and TPP1 seem to be promising target for radiosensitization (Table 3).

5.2.1TRF2

TRF2, one of the central proteins of the shelterin complex, is involved in the t-loop formation and in the initiation of the 3’-overhang invasion into duplex DNA [146]. TRF2 is substantial in end- protection through suppression of ATM kinase pathway or end-to-end fusions and takes part in the prevention of DNA damage machinery activation at telomeres [147,148]. It has been shown that TRF2 phosphorylation upon DNA damage is essential for DNA DSB repair in genomic as well as in telomeric DNA [148]. Complete silencing of the TRF2 protein is very detrimental for the cell and, as a consequence, only the effect of its partial silencing has been investigated. Partial suppression of TRF2 by siRNA in mesenchymal stem cells increased histone H2AX phosphorylation, senescence and reduced the surviving fraction in response to 2.5 Gy of γ-rays [149]. These results were supported by the evidence that ectopic low expression of TRF2 leads to telomere shortening and radiosensitization of radioresistant clones derived from telomerase-positive human lung A549 carcinoma cells as well as ALT-positive osteosarcoma cells U2OS [150].

5.2.2TPP1

TPP1 is another component of the shelterin complex that has been linked to IR sensitivity. TPP1 (also known as TINT1/ PTOP/PIP1) is a key member of the shelterin complex and participates to the formation of the core shelterin [1]. Indication of a role of TPP1 in the cellular response to IR

came from experiments in which TPP1 knockdown in MEFs activates an ATM-dependent DDR, which is characterized by the formation of TIFs [151] and is supported from evidence of TPP1 over-expression in radioresistant cells [62]. In particular, ectopic overexpression of TPP1 determines telomere lengthening, prolonged radiation-induced G2/M arrest, increased ATM and ATR expression and suppression of spontaneous TIF, thus conferring radioresistance to colorectal cancer cells [63]. Recently, Qiang and coworkers showed that TPP1 depletion induces apoptosis and caused enhanced radiation sensitivity in U2OS (ALT positive) cells. The molecular mechanism underlying the role of TPP1 in ALT cells remain to be clarified, however, the possibility to radiosensitize telomerase-positive and ALT-positive cancer cells trough TPP1 depletion makes this protein a very attractive target for the developing of new drug therapies [152].

5.3Other strategies

An alternative approach to activate telomere-based DDR consists in the use of telomere homolog oligonucleotides, termed T-oligos [153]. Although the detailed mechanism of tumor inhibition by T-oligos has not been completely elucidated, it is believed that the guanine (G)-rich T-oligos may favour the G4 formation in single-stranded telomeric DNA. As for G4 ligands, the final effect is the stall of the DNA replication forks and the promotion of DDR at telomeres that in turn lead to cellular senescence and apoptosis [154]. In single treatments, T-oligos have been shown to inhibit growth and induce apoptosis, autophagy, and/or senescence in a panel of human pancreatic, ovarian, breast cancer, melanoma, fibrosarcoma, and GBM [153,155-158]. Interestingly, they seem to be ineffective in normal tissues after either local or systemic administration in multiple mouse models [153,158,159] with a very low or absent toxicity and side effects.
T-oligos have been shown to rapidly concentrate in nuclei when added to cultured cells and the subsequent response require the WRN helicase [160], the protein mutated in the progeroid cancer- prone Werner syndrome. T-oligos and WRN interaction results in the formation of DNA -H2AX damage-like foci at the telomeres [160] with the consequent activation of ATM [157,158,161] and

its many downstream effectors, leading to apoptosis and senescence in human carcinoma cells [155,158]. Recently, it has been demonstrated that cells derived from mouse mammary tumors pre- treated with T-oligos and then exposed to -rays displayed a reduced cell growth and surviving fraction. Authors reported that the pretreatment with T-oligos (40 M for 24 hours) were able to reduce the surviving fraction to 2 Gy -rays from 0.72 to 0.31, in untreated and T-oligos treated cells, respectively. Moreover, cells exposed to coupled treatment displayed a delayed DSBs repair kinetic, undergone senescence, and died through apoptosis. Same results were obtained when treated cells were injected in the flanks of syngenic mice. Remarkably, combined treatment was able to reduce tumor growth of spontaneously occurring mammary carcinomas in transgenic mice [162].
Additional evidence regarding potential molecules able to render telomeres dysfunctional thus leading to radiosensitization arises from experiments using a compound named KML001 (i.e., sodium metarsenite, NaAsO2). Interestingly this compound has entered phase I clinical trial as single agent or in combination with Cisplatin for the treatment of advanced non-small cell lung cancer and other platinum responsive malignancies (see www.ClinicalTrials.gov). Despite the mechanism of action of this molecule is not fully understood, KML001 directly targets telomeres and induces the activation of the DNA damage signalling and rapid telomere erosion in prostate cancer cells [163]. KML001 determines a sensitization to the alkylating agent Temozolomide [164]
and to IR in a panel of GBM cells (U251MG, U138MG, U87MG and U373MG), as shown by surviving fraction experiments and in vivo evidence in orthotopic xenograft models [164].

6.T/TTCs as radiosensitizers: pros and cons

All the strategies listed in this review presented their own potential benefits and, on the other hand, also possible adverse effects. In general the principal merit of telomerase targeting is the great specificity for cancer cells due to the almost exclusive presence of re-activated telomerase. Considering as a tolerable risk the possible targeting of normal telomerase positive somatic cells,

the fascinating possibility to pursue a broad-spectrum cancer therapy fuelled the research over the past two decades.
Among the somatic cells that express telomerase, cells of the hematopoietic compartment are those that may greatly suffer telomerase inhibition due to telomerase activity in progenitor cells. However, normal hematopoietic stem cells should be less affected by telomerase inhibition therapy than neoplastic cells, which have a higher rate of proliferation and in general higher amount of telomerase activity. In addition, since telomeres of normal hematopoietic cells are likely to be longer than those in most advanced leukaemia, anti-telomerase therapy could be designed to end at a time when the tumor telomeres have been completely eroded while normal hematopoietic stem cells still have considerable telomeres remaining, opening a therapeutic window to inhibit telomerase in cancer cells sparing the stem cell compartment [165-167] and providing a difference in telomere length that could render IR treatment selectively more effective in cancer cells. On the other hand, targeting of telomerase present two major concerns. The first one regards the time required for telomerase inhibition that, as previously stated, is dependent on the initial telomere length in the tumor population. Different studies in cultured cells indicated that also in case of effective inhibition of telomerase activity cells continue to divide before cell death ensues, requiring from several weeks to months to obtain the block of proliferation [168]. This phenomenon has been attributed to the number of cell divisions required to reach a critical telomere length and a general problem is the quite long time that elapses between the first drug administration and the block of cell growth [169]. Moreover, in telomerase inhibitor and IR coupled treatments time will be prolonged by conventional RT treatments that for solid tumors deliver total doses of 60-80 Gy over almost two additional months. The second issue is represented by resistance mechanisms to telomerase inhibition. Inhibition of telomerase lead to progressive telomere shortening and when telomeres of cancer cells reach a critical length, a crisis phase arise. At this stage, a very strong selective pressure may favour the growth of resistant cells such as those that use a different telomere maintenance mechanism such as ALT [170-172]. ALT cancer cells are often less

aggressive than their telomerase-positive counterpart [173] whereas some reports indicate a higher radioresistance due to the interference of the ALT pathway with the DDR mechanism [174,175], indicating that telomerase inhibition may favour the relapse of ALT-positive/IR-resistant tumors. In recent years many efforts have been employed to find a “key protein” of ALT in order to develo p inhibitors of the ALT-mechanism. However, the development of ALT-targeted therapies is quite puzzling since, in contrast to telomerase, ALT has no a known specific enzymatic activity, and all the enzymes identified to date that play a role in ALT are also critical to normal cellular functions. Regarding the use of telomere targeting compounds there are same concerns in pursuing strategies that directly hamper the stability of telomeres. Indeed, telomeres are present in cancer as well in normal cells, and hence the risk of cytotoxicity in the course of such approaches is concrete. Data on the effects of telomere targeting in normal cells are available principally for G4 ligands. Surprisingly, data reported on literature reveal a resistance to telomere targeting higher in normal cells than in cancer cells [126,137]. This phenomenon could be related to the evidence that cells with longer telomeres (e.g. somatic normal cells) generally display a higher resistance to G4 ligands than cells with shorter telomeres (e.g. cancer cells) [133], indicating that telomere length may be the leverage of a possible therapeutic application. However, more than telomere length per se, the sum of different telomeric related factors such as telomere loss and telomeric proteins abundance and/or function may contribute to explain the different sensitivity of cancer and normal cells to G4 ligands. Another issue posed by the targeting of telomeres by G4 ligands resides in the evidence that even the most specific telomeric G4 ligand retains the ability to bind other G4 in gene promoters or in other regulatory regions of the genome. For instance, it is known that Telomestatin binds to G4 in promoters of gene such as c-Myb [176] and PDGFR-(platelet derived growth factor receptor )
[177] and that RHPS4 recognizes G4 in c-Myc [134], vegfr-2 [178] and CD133 genes [143].

However, it is not possible to rule out the possibility that also further genes may be transcriptionally inhibited by telomeric G4 ligands. Ignoring the target spectrum of a compound represent a serious problem and may pose some risk also in the combined treatments with IR. Indeed, the G4-

dependent targeting of genes involved in DDR would increase the severity of radiation-induced effects in normal cells that surround the tumor. Of note, putative unreported G4 motifs have been identified in the promoter of the ATM gene, the central kinase involved in the DDR [179]. Nevertheless, the pharmacological targeting of telomeres presents also strong positive features. Unlike telomerase inhibitors, telomere targeting agents do not require telomere shortening to be effective, but rather act through the induction of telomere dysfunction that in turn rapidly prompt DDR and cell cycle checkpoints activation, thus providing a more applicable tool in the perspective of RT. Moreover, the use of telomere targeting compounds seems to be effective in either telomerase positive or ALT positive tumors [132,180,181], as demonstrated for G4 ligands Telomestatin and RHPS4 and for siRNA mediated suppression of TRF2 and TPP1 [150,152].

7.Conclusions

RT is an essential tool in local control of many, if not most, cancer types and radiation resistance is one of the most frequent complications that radio-oncologists face in the treatment of solid tumors. In this context the development of new strategies to radiosensitize cancer cells is of crucial interest and among them telomerase/telomere targeting is one of the emerging and most promising approach.
However, much work is still required to ascertain the real potentiality of T/TTCs in combination with IR and, to date, only preclinical data are available in literature. Unfortunately, more than 30 years after the discovery of telomerase and of its role in telomere maintenance, an effective telomere/telomerase-based cancer therapy has yet to be approved for clinical use and currently trials testing radiosensitizing properties of T/TTCs have not been planned at all. However, it is undeniable that the study of the role played by telomeres in the radiation response has enlighten the tight crosslink between telomere function and both cellular and organismal sensitivity/resistance to IR opening new therapeutic routes that may yet greatly influence RT-based cancer treatment.

Acknowledgements:

We thank Dr. Alessandra di Masi for critical reading of the manuscript. The author’s research is supported by INFN Experiment RDH and IRPT.

Conflict of interest statement

The authors declare that there are no conflicts of interest

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Legend to figures

Figure 1 – Schematic representation of the telomeric-loop in the closed state, stabilized by shelterin proteins (i.e., TRF1, TRF2, RAP1, TIN2, TPP1, POT1) (a). Extensive telomeric erosion or telomere deprotection activates telomere dysfunction that results in several biological processes including (b) the DNA damage response, the block of cell cycle, and eventually cell death.

Figure 2 – Rationale for telomere and telomerase targeting strategies aimed to improve ionizing radiation response. Telomere and telomerase targeting cause the activation of different cellular responses converging to telomere dysfunction that in turn radiosensitize cancer cells.

Figure 3 – Schematic summary of the strategies developed to radiosensitize cancer cells. (a) Inhibition of the telomerase and (b) targeting of the telomere structure.

Table 1. DDR protein involved in telomere maintenance

Protein
Species
Function
Sensitivity
Telomeric effects Length Fusions
Shelterin interaction/Shelterin
protein involved
Reference

Ku70/80 Mouse; Human NHEJ IR, Ox Shorter Yes Yes/TRF1, TRF2, RAP1 [182-184]
DNA-PKcs Mouse NHEJ IR,Ox Shorter/longer Yes Yes/TRF2 (KIP mediated) [185,186]
XRCC4 Mouse NHEJ IR ND Yes ND [187]
Artemis Mouse; Human NHEJ IR ND/Shorter Yes ND [188,189]
Rad54 Mouse HR IR Shorter Yes ND [190]
RAD51D Mouse; Human HR IR Shorter Yes Y/ND [191]
Brca1 Mouse HR IR Shorter Yes Yes/TRF1, TRF2 [192,193]
MUS81 Mouse; Human HR ND ND ND Yes/TRF2 [194]
MRE11 Human DSBs sensing IR Normal ND Yes/RAP1 [184,195]
NBN Human DSBs sensing IR Normal/shorter No Yes/TRF1, TRF2 [30,195-197]
ATM Mouse; Human DSBs sensing IR Shorter Yes Yes/ TRF1, TR2 [182,198,199]
RAD50 Human DSBs sensing IR, UV, Ox Normal Yes Yes/TRF1, TRF2, RAP1 [184,195,200,201]
Rad9/Hus1/Rad1 Mouse DSBs sensing IR Shorter Yes Yes/ND [202]
DNA Ligase III Human BER; DSBs repair ND ND ND Yes/TRF1, POT1 [200]
APEX1 Human BER; DSBs repair ND ND ND Yes/TRF1, TRF2, RAP1 [203]
PARP1 Human BER IR, Ox Shorter Yes Yes/TRF2, RAP1 [184,204,205]
PARP2 Human BER IR, Ox Normal No Yes/TRF2 [206]
Polb Mouse BER ND Longer Yes Yes/TRF2 [207]
MPG Human BER ND ND ND Yes/RAP1 [203]
ERCC1 Mouse; Human NER UV Normal No ND [208]
DDB1 Human NER ND ND ND TRF1, TRF2 [200]
POLD1 Human NER; MMR ND ND ND Yes/TRF2, RAP1 [200,203]
FEN1 Human Nucleases ND ND ND Yes/TRF2 [203]
WRN Human DNA unwinding ND ND ND Yes/TRF2; POT1 [209,210]
BLM Human DNA unwinding ND ND ND Yes/TRF1, TRF2, POT1 [210,211]

RECQL4
Human
DNA unwinding
ND
ND
ND
Yes/TRF1, TRF2, TPP1, RAP1,
POT1
[203,212]

RNF8 Human Ubiquitination ND Shorter Yes Yes/TPP1 [213]

NHEJ: Non homologous end joining; HR: Homologous recombination; BER: Base excision repair; NER: Nucleotide excision repair; MMR: mismatch repair; IR: Ionizing radiation; Ox: Oxidative stress; UV: Ultraviolet light; ND: Not Determined.

Table 2. Summary of the relevant findings on telomerase inhibition and IR coupled treatment in human tumor in vitro and in vivo models

Telomerase inhibition
strategy
IR type and dose range
Experimental model
Biological effects of combined treatment
Reference

TERT haploinsufficiency
Not specified (2-8 Gy)
HCT116 (colon cancer cells)
Reduction of clonogenic survival and increase of SA-
gal positive cells
[69]

TERT siRNA
Not specified (2-6 Gy)
HeLa and SiHa
(uterine cervical cancer cells)
Reduced colony-forming efficiency
[40]

Survivin promoter-driven
TERT siRNA
X-rays (1-7 Gy)
HeLa cells
Reduced colony-forming efficiency
[72]

TERC siRNA
X-rays (2-8 Gy)
HeLa cells in vitro and HeLa
derived eterotopic mouse xenograft
Reduced colony forming efficiency and decreased growth of tumor volume in vivo
[73]

TERT siRNA X-rays (2-8 Gy) SiHa cells Reduced colony forming efficiency [74]

AS-ODN complementary
to bases 38-50 of TERC
Gamma rays (1-10 Gy)
U251 (glioma cells)
Reduction of clonogenic survival and increased IR-
induced DNA damage over the first 6h
[82]

GRN163L
(IMETELSTAT)
X-rays (2-7 Gy)
MDA-MB-231 (breast cancer cells) and eterotopic xenograft
mouse model
Reduction in surviving fraction at 2 and 4 Gy in single
dose exposition and at 4 Gy (2×2 Gy) in fractionated
dose exposition; reduction in tumor growth rate in
nude mice
[86]

GRN163L
(IMETELSTAT)
X-rays
(fractionated; 5×2 Gy)
Orthothopic xenograft mouse
model from U87MG cells
Reduction in tumor volume and increase in mice
survival after fractionated dose exposition
[95]

EGCg
X-rays (0-30 Gy)
Human brain microvascular endothelial cells (HBMEC)
Reduction of cell growth, induction of cicline kinase
inhibitors p21 and p27, induction of apoptosis and
necrosis.
[109]

EGCg
X-rays (ND)
HeLa, K-562 (chronic
myelogenous leukemia) and IM-
9 (multiple myeloma) cells
Enhancement of apoptosis and decrease in cell proliferation
[110]

MST-312
X-rays (2 Gy)
HepG2 (Hepatoma cells)
Reduced colony forming efficiency, increased p53
expression, decreased mitochondrial membrane
potential and augmented apoptosis frequency
[111]

AZT
Gamma rays (1-10 Gy)
U251 cells
Decreased DNA strand breaks repair rate, reduced
colony forming efficiency
[119]

Table 3. Summary of the relevant findings on telomere targeting compounds and IR coupled treatment in human tumour in vitro and in vivo models

Telomere targeting
strategy
IR type and dose
range
Experimental model
Biological effects of combined treatment
Reference

TAC (G4 ligand)
X-rays (2-8 Gy)
SF763 and SF767 glioma cells
Reduction of clonogenic survival, delayed DNA DSBs repair kinetic,
accumulation in G2-M pahse, increased frequency of chromosomal
aberrations
[144]

RHPS4 (G4 ligand)
X-rays (0.5-6 Gy)
U251MG, U87MG, T67 and T70 glioma cells, AG01522 and MRC5 primary fibroblasts
Reduced long term growth and surviving fraction, block of cell cycle and
accumulation in G2 phase, delayed DSBs repair kinetic and increase in telomere-related chromosomal aberrations
[137]

Pt-Ctpy (G4 ligand)
X-rays (2-8 Gy) SF763, SF767; A549, and H1299 (non-small cell lung cancer) and SF763 derived mouse xenograft
model
Reduced clonogenic survival, delayed DNA DSBs repair kinetic, increased TIF frequency and telomere loss, delayed tumor growth rate
and increased mice survival
[145]

Partial TRF2
knockdown by siRNA
Gamma rays
(2.5 Gy)
hMSC cells, mesenchimal stem cells
Increased fraction of senescent cells, reduced colony forming efficiency
[149]

TRF2 knockdown by
siRNA
X-rays (2-10 Gy) A549 lung carcinoma and U2OS
osteosarcoma cells
Reduced surviving fraction
[150]

POT1 knockdown by
siRNA
Gamma rays
(1-10 Gy)
Hep-2 larynx squamous carcinoma
Reduced surviving fraction
[58]

TPP1 Knockdown by
siRNA
X-rays (1-10 Gy)
U2OS
Reduced surviving fraction
[152]

T-oligo
Gamma rays
(2-12 Gy)
Primary mammary tumor cells derived from transgenic mice that spontaneously develop mammary
carcinoma and in vivo mouse model
Reduced cell growth rate and surviving fraction, delayed DSBs repair kinetic, increased senescence and apoptosis as evaluated in vitro as well
as in vivo and reduced tumor growth in vivo
[162]
BIBR 1532
KML001
Not specified (1-9
Gy)
U251MG, U138MG, U87MG and U373MG glioblastoma cell
lines and orthotopic xenograft
mouse model
Reduced cell survival, increased DNA damage and apoptosis, decreased tumor volume in the orthotopic mouse model
[164]