The Relevance of Telomerase and Telomere-Associated Proteins in B-Acute Lymphoblastic Leukemia

Telomeres and telomerase are closely linked to uncontrolled cellular proliferation, immortalization and carcinogenesis. Telomerase has been largely studied in the context of cancer, including leukemias. Deregulation of human telomerase gene hTERT is a well-established step in leukemia development. B-acute lymphoblastic leukemia (B-ALL) recovery rates exceed 90% in children; however, the relapse rate is around 20% among treated patients, and 10% of these are still incurable. This review highlights the biological and clinical relevance of telomerase for B-ALL and the implications of its canonical and non-canonical action on signaling pathways in the context of disease and treatment. The physiological role of telomerase in lymphocytes makes the study of its biomarker potential a great challenge. Nevertheless, many works have demonstrated that high telomerase activity or hTERT expression, as well as short telomeres, correlate with poor prognosis in B-ALL. Telomerase and related proteins have been proven to be promising pharmacological targets. Likewise, combined therapy with telomerase inhibitors may turn out to be an alternative strategy for B-ALL.


Introduction
Leukemia is characterized by the production of abnormal leukocytes based on cytogenetic alterations, molecular modifications, clinical features and, notably, high proliferation [1]. Although knowledge on molecular alterations that lead to leukemogenesis as well as in the mechanisms involved in disease maintenance and propagation has gradually increased over the years, the identification of novel strategies to treat the disease is still lacking. Telomeres and telomerase are closely associated with cell proliferation, which makes them attractive targets for studies in oncology [2]. Telomerase is a reverse transcriptase that elongates the telomeres, thereby compensating the loss of telomere repeats after successive replication cycles; this is a phenomenon integrated to carcinogenesis known as cell immortalization [3]. Therefore, telomerase activity (TA) is detectable in almost all types of malignant cells, including leukemia cells. In this article, we discuss the relevance of telomerase and other telomere-associated factors to B-acute lymphoblastic leukemia (B-ALL) as well as their implications for development of new treatments, highlighting the role of these proteins as potential markers of B-ALL.

Materials and Methods
We conducted a literature search using the NCBI database (PubMed) in September 2022 using the following combinations of keywords: ("telomerase" OR "hTERT") AND ("Acute lymphoblastic leukemia" OR "B-ALL" OR "ALL-B" OR "ALL") and ("telomerase" OR "hTERT") AND ("Acute lymphoblastic leukemia") AND "treatment". Additional references were obtained from cross-referencing bibliographies.
The telomeric tandem repeat sequences of TTAGGG are located in the end of chromosomes. They are bound by a specialized protein complex known as shelterin [48]. The telomeres play vital roles in cellular processes due to their capacity to protect chromosomes from end-to-end fusions and genome instability [49,50]. Cells with absent telomere maintenance mechanisms exhibit a maximum cell division capacity. Due to the loss of chromosome-capping function in telomeres, the cell enters senescence or is lead to apoptosis [51,52]. Furthermore, G-rich telomere repeat sequences are susceptible to oxidative damage, which reinforces telomere shortening and leads to cell senescence related to aging [53].
The shelterin complex is composed of six protein subunits-TRF1, TRF2, RAP1, TIN2, ACD, and POT1 ( Figure 1) [54]. Although shelterin have many functions, such as protecting the telomeres from DNA deterioration and preventing activation of unwanted repair systems, they also play a key role in telomerase activity regulation [55][56][57].
Shelterin are also involved in the establishment of heterochromatin and telomeric silencing. The recruitment of these proteins is apparently related to enriching methyltransferases into the sub-telomere regions for gene silencing, which allows telomere lengthening [58].
The composition of white blood cells depends on different exposures to stress factors [59]. Different stressors can initiate a redistribution of leukocytes from immune reservoirs to the circulation. This is relevant due the fact that telomere length (TL) differs among leukocyte subtypes (lymphocytes, monocytes, granulocytes). Moreover, naïve leukocytes have telomeres similar to those found in hematopoietic stem cell progenitors, while smaller telomeres are present in mature leukocytes. This makes it difficult to define if alterations in TL can be attributed to a blood sample leukocyte composition or to a particular condition [60,61].

Figure 1.
Telomerase representation. Dyskerin complex (NHP2, NOP10 and DKC) binds to hTR through its ACA domain. TCAB1 binds to TERT and to hTR. The template region of hTR binds to the telomeric 3′ -end strand. Shelterin binds to telomeric repeat region. TRF2 interacts with RAP1 and TRF1, binding directly to the telomeric DNA and to TIN2, which also binds to TPP1. TPP1 interacts with POT1, which is responsible for recruiting telomerase to telomeres through the TEN domain of TERT.
Shelterin are also involved in the establishment of heterochromatin and telomeric silencing. The recruitment of these proteins is apparently related to enriching methyltransferases into the sub-telomere regions for gene silencing, which allows telomere lengthening [58].
The composition of white blood cells depends on different exposures to stress factors [59]. Different stressors can initiate a redistribution of leukocytes from immune reservoirs to the circulation. This is relevant due the fact that telomere length (TL) differs among leukocyte subtypes (lymphocytes, monocytes, granulocytes). Moreover, naïve leukocytes have telomeres similar to those found in hematopoietic stem cell progenitors, while smaller telomeres are present in mature leukocytes. This makes it difficult to define if alterations in TL can be attributed to a blood sample leukocyte composition or to a particular condition [60,61].

Telomerase and Cancer
The telomerase consists of the catalytic telomerase reverse transcriptase subunit, known as TERT, and an RNA component (hTR) that works as a template for telomere extension [62][63][64]. The canonical functions of telomerase are related to telomere length maintenance and genome stability, while the non-canonical functions are involved in the regulation of non-telomeric DNA, alterations in cell cycle kinetics, the rise of proliferation, chromatin remodeling and more (Table S1) [65][66][67].
Telomerase acts on TL maintenance during the fetal phase of life, and its presence in adult tissues is infrequent. In leukocytes, TL is stabilized around age 20, and a slow rate of attrition occurs during adulthood [68]. Moreover, strong telomerase activity is found in progenitor stem cells and activated lymphocytes, and it is especially enhanced in carcinogenesis, with implications for genome integrity, proliferation and stemness [69].
Telomerase is present in tumor cells from over 85% of cancer types, while about 15% of them continue the telomere lengthening through homologous recombination processes collectively known as alternative lengthening of telomeres (ALT), which is not a telomerase-dependent mechanism [70][71][72].
The regulation of telomerase activity is crucial and occurs mainly through the control of hTERT transcription, which also determines in which type of cell telomerase will be Figure 1. Telomerase representation. Dyskerin complex (NHP2, NOP10 and DKC) binds to hTR through its ACA domain. TCAB1 binds to TERT and to hTR. The template region of hTR binds to the telomeric 3 -end strand. Shelterin binds to telomeric repeat region. TRF2 interacts with RAP1 and TRF1, binding directly to the telomeric DNA and to TIN2, which also binds to TPP1. TPP1 interacts with POT1, which is responsible for recruiting telomerase to telomeres through the TEN domain of TERT.

Telomerase and Cancer
The telomerase consists of the catalytic telomerase reverse transcriptase subunit, known as TERT, and an RNA component (hTR) that works as a template for telomere extension [62][63][64]. The canonical functions of telomerase are related to telomere length maintenance and genome stability, while the non-canonical functions are involved in the regulation of non-telomeric DNA, alterations in cell cycle kinetics, the rise of proliferation, chromatin remodeling and more (Table S1) [65][66][67].
Telomerase acts on TL maintenance during the fetal phase of life, and its presence in adult tissues is infrequent. In leukocytes, TL is stabilized around age 20, and a slow rate of attrition occurs during adulthood [68]. Moreover, strong telomerase activity is found in progenitor stem cells and activated lymphocytes, and it is especially enhanced in carcinogenesis, with implications for genome integrity, proliferation and stemness [69].
Telomerase is present in tumor cells from over 85% of cancer types, while about 15% of them continue the telomere lengthening through homologous recombination processes collectively known as alternative lengthening of telomeres (ALT), which is not a telomerasedependent mechanism [70][71][72].
The regulation of telomerase activity is crucial and occurs mainly through the control of hTERT transcription, which also determines in which type of cell telomerase will be expressed [62]. The regulation of the active enzyme, on the other hand, is performed by a post-transcriptional maturation process involving binding to hTR (which is constantly expressed) [48]. Moreover, the regions containing TERT and hTR genes, 5p15.33 and 3q26.3, respectively, are usually amplified in cancer cells [73]. However, the hTERT mutation itself has been shown to be insufficient for telomere maintenance [74].
Mutations in the hTERT promoter represent frequent somatic genetic alterations that cause telomerase reactivation [75,76]. Epigenetic changes are also involved in different steps of this reactivation, including DNA methylation of hTERT controllers that are associated with transcription activators such as c-myc, MZF-2, and WT-1. Hypermethylation prevents binding of the repressors to the promoter, which leads to hTERT upregulation and telomerase activation [77,78].
Telomerase was first described for its capacity to elongate telomeres [79]. Nevertheless, it is becoming clear that TERT is also involved in distinct biological pathways [80] that are related to both physiological and pathological processes. These processes include those that contain stem cell functions, homeostasis, aging, tumor progression, drug resistance, regulation of non-telomeric DNA damage responses, promotion of cell growth and proliferation, acceleration of the cell cycle and damage to mitochondrial DNA, which influences cell integrity following oxidative stress. For these non-canonical activities, telomerase was reported to act on the activation of the senescence signaling pathway, the induction of apoptosis through mitochondrial pathways, autophagy, cellular growth, NF-kB mediated inflammation, and cancer progression in general (Table S1).
Telomerase expression and activity are also influenced by factors from distinct pathways. P23, for example, acts as an anti-apoptotic factor that plays a significant role in estrogen receptor α signal transduction, but which can also regulate TA by binding directly to the catalytic subunit of telomerase. This interaction is required for TL maintenance for an efficient telomerase assembly, helping to modulate telomerase-DNA binding in extension activities. Thus, the overexpression of p23 causes B-ALL cells to evade apoptosis for both TERT-related and independent pathways [81]. Similarly, c-MYC promotes hTERT deregulation, resulting in the reduction of telomere length, telomerase activity and cell proliferation [82]. Additionally, TERT can act as a transcription co-factor that regulates expression of several genes [77], which are summarized in Supplementary Table S1 .
Interestingly, there is evidence that TA is also related to gender. Male Egyptian B-ALL patients, for example, were reported to have higher expression and activity of TERT than female patients. In this particular study, the total leukocyte count of both groups of patients was higher when telomerase is upregulated, indicating a poor response to therapy [166].
The multi-functional profile of telomerase, as well as its relevance for carcinogenesis and cancer maintenance, make it an extremely relevant target for the development of studies focusing on both therapeutical purposes and on the understanding of tumor biology, especially in blood-related diseases [167][168][169].

Telomerses and Telomerase in B-Acute lymphoblastic leukemia
The hTERT mRNA can be detected in memory and naïve germinal center B-cells (GC) in which its level is associated with high TA [170,171]. The expression of telomerase in GC B-cells is inducible during immunological response, but at lower levels than in leukemic cells [172]. The synergistic stimulation with anti-IgM Ab plus specific cytokines (IL-2, IL-4, and IL-13), as well as the surface molecules BCR or CD40, increase telomerase activity in B-cells. Then, the canonical telomerase functions seem to be the main mechanism for telomere length maintenance in the germinal center in normal (non-pathological) conditions ( Figure 2).
Despite the fact that up-regulation of telomerase in human B lymphocytes may occur independently of cellular proliferation, with expression of telomerase catalytic subunits [173], it has been demonstrated that telomerase activity can also be induced by PI-3 kinase-dependent and independent pathways linked to proliferation. For instance, the inhibition of PI3K blocked the anti-IgM plus anti-CD40-induced telomerase expression in B cells in a dose-dependent manner [172,174].
Telomerase is virtually absent in most adult tissues and detectable in most tumors, but the physiological role of this enzyme in lymphocytes represents an important challenge for approaching it as biological marker in leukemia. However, the straight relationship between telomerase activity and proliferation, as well as its anti-apoptotic role [175], make it essential for leukemogenesis. The uncontrolled proliferation of B lymphoblastic precursor cells in B-ALL leads to shortened telomeres and raised telomerase activity [176,177]. Additionally, leukemia cells with a normal karyotype exhibit longer telomeres when compared with cells with abnormal karyotypes [176,177].
Both high telomerase activity and shortened telomeres are correlated with disease progression, resistance to therapy and bad prognosis in ALL [178]. Telomerase can block apoptosis mechanisms in leukemic blasts, resulting in faster disease progression, and its activities are related with lactate dehydrogenase, which is an unfavorable prognostic factor for ALL patients [166]. In this sense, recent studies have revealed telomerase overexpression and hTERT methylation status as a promising prognostic biomarkers in B-ALL (especially for childhood disease), and more precisely for maintenance and disease persistence, also reinforcing the potential of telomerase as therapeutical target, mainly due to its multiple non-canonical actions [45,47,179].
In Ph+ B-ALL, the p16INK4A/pRb pathway with a high TA determines a group of adult ALL associated with poor prognosis [180]. Furthermore, Philadelphia chromosome genes also regulate telomerase and its activity at multiple levels [181]. Antisense Inhibition of BCR/ABL, for example, is able to enhance telomerase activity, leading to activation of tyrosine kinase proteins and inhibition of apoptosis [25,26].  Telomerase is virtually absent in most adult tissues and detectable in most tumors but the physiological role of this enzyme in lymphocytes represents an important chal lenge for approaching it as biological marker in leukemia. However, the straight relation ship between telomerase activity and proliferation, as well as its anti-apoptotic role [175] make it essential for leukemogenesis. The uncontrolled proliferation of B lymphoblasti precursor cells in B-ALL leads to shortened telomeres and raised telomerase activity [176,177]. Additionally, leukemia cells with a normal karyotype exhibit longer telomere when compared with cells with abnormal karyotypes [176,177].
Both high telomerase activity and shortened telomeres are correlated with diseas progression, resistance to therapy and bad prognosis in ALL [178]. Telomerase can block apoptosis mechanisms in leukemic blasts, resulting in faster disease progression, and it activities are related with lactate dehydrogenase, which is an unfavorable prognosti factor for ALL patients [166]. In this sense, recent studies have revealed telomeras overexpression and hTERT methylation status as a promising prognostic biomarkers in B ALL (especially for childhood disease), and more precisely for maintenance and diseas persistence, also reinforcing the potential of telomerase as therapeutical target, mainly du However, controversial results can be found in the literature. In the work of Ozgur et al. [182] and Eskandari et al. [179], no significant association was found between hTERT mRNA expression and hematological parameters in B-ALL. Nevertheless, these same studies have showed that telomere attrition is linked to childhood ALL. On the other hand, Borssén et al. have demonstrated that B-cell precursor group cases had a higher hTERT methylation than diploid ALL. In addition, hTERT mRNA levels were negatively associated with methylation status, but curiously, in low-risk B-cell precursor patients, long telomeres indicated a worse prognosis [183].
Monitoring minimal residual disease (MRD) is one of the most important strategies to follow up B-ALL patients due the capacity to identify lower cell levels. In this sense, it was demonstrated that quantification of telomerase expression along with monitoring MRD by qPCR can strengthen the follow up of patients with B-ALL. This would not only improve treatment follow up but also help to identify post-therapy remission [47].
Finally, a large number of studies propose that pathogenesis and the phenotypic characteristics of B-acute lymphoblastic leukemia are connected with the conjunction of specific targets and DNA variations promoted by epigenetic alterations such as methylation [184][185][186]. hTERT promoter methylation is infrequent in B-ALL cases with remission, and there is no association with TL. However, hTERT RNA expression is reduced when methylation occurs [183]. Methylation of CDKN2B CpG island was associated with high telomerase activity in children with B-ALL [187]. It has also been shown that β-Arrestin1 promotes cellular senescence in B-ALL by binding with P300-Sp1 in order to regulate hTERT transcription. In that case, hTERT is a major factor due to the regulation stimulated on the β-Arrestin pathway, rising p300-sp1 expression [87].

Shelterin in B Lymphoblastic Leukemia
The role of the shelterin complex in B-ALL has also been studied. TRF2 expression was shown to be increasing in acute leukemias and also higher in lymphocytes of B-ALL patients, particularly in those with an abnormal karyotype [177]. Recently, NOTCH3, PAX5, CBFB, and particularly ACD were shown to drive the activated RAS pathway and monosomy 7 to B-acute lymphoblastic leukemia [188]. Nonetheless, ACD plays a key role in telomere maintenance due to its interaction with POT1; this combination protects telomeres and recruit telomerase at chromosome ends. Despite the overexpression of wild-type, ACD does not lead to telomere lengthening, the G223V mutation reflects on TL and seems to be related to decreased apoptosis activity in B-ALL cells, that is triggered to the functional role of ACD and its relevance for cell survival in leukemia [189].
Beyond hTERT, B-ALL patients also show high expression of CTC1 and OBFC1 (they are part of CST complex which works with the shelterin complex to lengthen telomeres); however, only CTC1 was associated with leukemia [190].

Telomerase and Genetic Variation
There is, currently, a vast field literature on hTERT polymorphisms and their implications in oncology, but just few works approach it in the context of B-ALL. The hTERT polymorphisms rs2735940 and rs2736100, for example, were defined as risk factor for ALL and turned out to be functional; they were implicated in TA, TL and homeostasis. The same authors showed that a variant near hTR, as well as high TL, are markers for risk of acute lymphoblastic leukemia in Chinese children [191].
Another study demonstrated that the survival rate of children with B-ALL was higher in European American children (EA) than in African American children (AA), which appeared to be due to the different canonical pathways affected in each case. Telomerase signaling is related to AA pathways, while chromosome aberrations in EA more frequently affect genes involved with homologous recombination [192]. This suggests that hTERT may have a different influence on B-ALL with regard to different populations; nonetheless, large-scale studies need to be done to verify this hypothesis.

Current Telomerase Inhibitors and Their Clinical Potential
Different approaches for telomerase inhibition have been under development for more than a decade, aiming at more effective treatment strategies. Telomerase activity can be inhibited by different strategies, such as disrupting biosynthesis, maturation, assembly, or correct interaction between the telomerase complex and the substrate [193]. In Table 1, we exemplified some of the currently available telomerase inhibitors.

Current Telomerase Inhibitors and Their Clinical Potential
Different approaches for telomerase inhibition have been under development for more than a decade, aiming at more effective treatment strategies. Telomerase activity can be inhibited by different strategies, such as disrupting biosynthesis, maturation, assembly, or correct interaction between the telomerase complex and the substrate [193]. In Table 1, we exemplified some of the currently available telomerase inhibitors.  [194][195][196] Zataria multiflora extract (ZME) Chemical structures of main volatile and nonvolatile constituents are in Sajed, Sahebkar and Iranshahi works [197] Combined with doxorubicin Pulmonoprotective action of Zataria multiflora ethanolic extract on cyclophosphamide-induced oxidative lung toxicity in mice Anti-leukemic effect of Zataria multiflora extract in combination with doxorubicin to combat acute lymphoblastic leukemia cells. [198,199]

2-[(E)-3naphtalen-2-ylbut-2enoylamino]benzoic acid (BIBR1532)
Monotherapy and combined with doxorubicin BIBR1532 exerts a series of anti-cancer activities linked to the inhibition of the canonical telomerase pathway and the TERT extra-telomeric functions in feline oral squamous cell carcinoma. BIBR1532 exhibits a selective cytotoxicity against primary leukemia cells from acute myeloid leukemia and chronic lymphocytic leukemia patients. Telomerase inhibition by BIBR1532 causes rapid cell death in pre-B-acute lymphoblastic leukemia cells BIBR1532 exerted potent cytotoxic effects on a panel of human cancer cells in a dose-dependent manner in leukemic cells which were more sensitive to the inhibitor BIBR 1532, exerts a direct short-term growth suppressive effect in a concentration-dependent manner possibly through the downregulation of c-Myc and hTERT expression [46,[200][201][202][203] lipidconjugated N30 -P50 thiophosphora midate GRN163L (Imetelstat)

Monotherapy
Imetelstat induces leukemia stem cell death in pediatric acute myeloid leukemia. The telomerase antagonist imetelstat efficiently targets glioblastoma tumor-initiating cells leading to decreased proliferation and tumor growth The inhibition of telomerase with imetelstat ex vivo led to significant dose-dependent apoptosis of B-ALL cells. Thus, imeteostat can be usefull in the standard treatment of B-ALL [45,204,205] Doxorubicin (DOX) is a chemotherapy drug used in different cancer treatment protocols. This molecule promotes cell death through disruption of DNA repair by inhibiting topoisomerase II, and provokes oxidative stress by generating free radicals [7,206]. However, doxorubicin has a high toxicity for the heart, which can lead to mortality among cancer patients, limiting its clinical applications [207].  [194][195][196] Zataria multiflora extract (ZME) Chemical structures of main volatile and non-volatile constituents are in Sajed, Sahebkar and Iranshahi works [197] Combined with doxorubicin Pulmonoprotective action of Zataria multiflora ethanolic extract on cyclophosphamide-induced oxidative lung toxicity in mice Anti-leukemic effect of Zataria multiflora extract in combination with doxorubicin to combat acute lymphoblastic leukemia cells. [198,199]

Current Telomerase Inhibitors and Their Clinical Potential
Different approaches for telomerase inhibition have been under development for more than a decade, aiming at more effective treatment strategies. Telomerase activity can be inhibited by different strategies, such as disrupting biosynthesis, maturation, assembly, or correct interaction between the telomerase complex and the substrate [193]. In Table 1, we exemplified some of the currently available telomerase inhibitors. Combined with doxorubicin MST-312 inhibits the progress of multiple myeloma by inhibiting the telomerase activity of this cells. Monotherapy long-term exposure to the MST-312 in U251 cells resulted in the induction of cell adaptations with possible negative clinical implications. MST-312 alters telomere dynamics, gene expression profiles and growth in human breast cancer cells [194][195][196] Zataria multiflora extract (ZME) Chemical structures of main volatile and nonvolatile constituents are in Sajed, Sahebkar and Iranshahi works [197] Combined with doxorubicin Pulmonoprotective action of Zataria multiflora ethanolic extract on cyclophosphamide-induced oxidative lung toxicity in mice Anti-leukemic effect of Zataria multiflora extract in combination with doxorubicin to combat acute lymphoblastic leukemia cells. [198,199] 2-[(E)-3naphtalen-2-ylbut-2enoylamino]benzoic acid (BIBR1532) Monotherapy and combined with doxorubicin BIBR1532 exerts a series of anti-cancer activities linked to the inhibition of the canonical telomerase pathway and the TERT extra-telomeric functions in feline oral squamous cell carcinoma. BIBR1532 exhibits a selective cytotoxicity against primary leukemia cells from acute myeloid leukemia and chronic lymphocytic leukemia patients. Telomerase inhibition by BIBR1532 causes rapid cell death in pre-B-acute lymphoblastic leukemia cells BIBR1532 exerted potent cytotoxic effects on a panel of human cancer cells in a dose-dependent manner in leukemic cells which were more sensitive to the inhibitor BIBR 1532, exerts a direct short-term growth suppressive effect in a concentration-dependent manner possibly through the downregulation of c-Myc and hTERT expression [46,[200][201][202][203] lipidconjugated N30 -P50 thiophosphora midate GRN163L (Imetelstat)

Monotherapy
Imetelstat induces leukemia stem cell death in pediatric acute myeloid leukemia. The telomerase antagonist imetelstat efficiently targets glioblastoma tumor-initiating cells leading to decreased proliferation and tumor growth The inhibition of telomerase with imetelstat ex vivo led to significant dose-dependent apoptosis of B-ALL cells. Thus, imeteostat can be usefull in the standard treatment of B-ALL [45,204,205] Doxorubicin (DOX) is a chemotherapy drug used in different cancer treatment protocols. This molecule promotes cell death through disruption of DNA repair by inhibiting topoisomerase II, and provokes oxidative stress by generating free radicals [7,206]. However, doxorubicin has a high toxicity for the heart, which can lead to mortality among cancer patients, limiting its clinical applications [207].

Monotherapy and combined with doxorubicin
BIBR1532 exerts a series of anti-cancer activities linked to the inhibition of the canonical telomerase pathway and the TERT extra-telomeric functions in feline oral squamous cell carcinoma. BIBR1532 exhibits a selective cytotoxicity against primary leukemia cells from acute myeloid leukemia and chronic lymphocytic leukemia patients. Telomerase inhibition by BIBR1532 causes rapid cell death in pre-B-acute lymphoblastic leukemia cells BIBR1532 exerted potent cytotoxic effects on a panel of human cancer cells in a dose-dependent manner in leukemic cells which were more sensitive to the inhibitor BIBR 1532, exerts a direct short-term growth suppressive effect in a concentration-dependent manner possibly through the downregulation of c-Myc and hTERT expression [46,[200][201][202][203] lipid-conjugated N30-P50 thiophosphoramidate GRN163L (Imetelstat) Genes 2023, 14, x FOR PEER REVIEW 8 of 20

Current Telomerase Inhibitors and Their Clinical Potential
Different approaches for telomerase inhibition have been under development for more than a decade, aiming at more effective treatment strategies. Telomerase activity can be inhibited by different strategies, such as disrupting biosynthesis, maturation, assembly, or correct interaction between the telomerase complex and the substrate [193]. In Table 1, we exemplified some of the currently available telomerase inhibitors.  [194][195][196] Zataria multiflora extract (ZME) Chemical structures of main volatile and nonvolatile constituents are in Sajed, Sahebkar and Iranshahi works [197] Combined with doxorubicin Pulmonoprotective action of Zataria multiflora ethanolic extract on cyclophosphamide-induced oxidative lung toxicity in mice Anti-leukemic effect of Zataria multiflora extract in combination with doxorubicin to combat acute lymphoblastic leukemia cells. [198,199] 2-[(E)-3naphtalen-2-ylbut-2enoylamino]benzoic acid (BIBR1532) Monotherapy and combined with doxorubicin BIBR1532 exerts a series of anti-cancer activities linked to the inhibition of the canonical telomerase pathway and the TERT extra-telomeric functions in feline oral squamous cell carcinoma. BIBR1532 exhibits a selective cytotoxicity against primary leukemia cells from acute myeloid leukemia and chronic lymphocytic leukemia patients. Telomerase inhibition by BIBR1532 causes rapid cell death in pre-B-acute lymphoblastic leukemia cells BIBR1532 exerted potent cytotoxic effects on a panel of human cancer cells in a dose-dependent manner in leukemic cells which were more sensitive to the inhibitor BIBR 1532, exerts a direct short-term growth suppressive effect in a concentration-dependent manner possibly through the downregulation of c-Myc and hTERT expression [46,[200][201][202][203] lipidconjugated N30 -P50 thiophosphora midate GRN163L (Imetelstat)

Monotherapy
Imetelstat induces leukemia stem cell death in pediatric acute myeloid leukemia. The telomerase antagonist imetelstat efficiently targets glioblastoma tumor-initiating cells leading to decreased proliferation and tumor growth The inhibition of telomerase with imetelstat ex vivo led to significant dose-dependent apoptosis of B-ALL cells. Thus, imeteostat can be usefull in the standard treatment of B-ALL [45,204,205] Doxorubicin (DOX) is a chemotherapy drug used in different cancer treatment protocols. This molecule promotes cell death through disruption of DNA repair by inhibiting topoisomerase II, and provokes oxidative stress by generating free radicals [7,206]. However, doxorubicin has a high toxicity for the heart, which can lead to mortality among cancer patients, limiting its clinical applications [207].

Monotherapy
Imetelstat induces leukemia stem cell death in pediatric acute myeloid leukemia. The telomerase antagonist imetelstat efficiently targets glioblastoma tumor-initiating cells leading to decreased proliferation and tumor growth The inhibition of telomerase with imetelstat ex vivo led to significant dose-dependent apoptosis of B-ALL cells. Thus, imeteostat can be usefull in the standard treatment of B-ALL [45,204,205] Doxorubicin (DOX) is a chemotherapy drug used in different cancer treatment protocols. This molecule promotes cell death through disruption of DNA repair by inhibiting topoisomerase II, and provokes oxidative stress by generating free radicals [7,206]. However, doxorubicin has a high toxicity for the heart, which can lead to mortality among cancer patients, limiting its clinical applications [207].
The Zataria multiflora extract (ZME) is a plant extract oil that exhibited a synergistic effect in association with doxorubicin, increasing its toxicity in all tested B-ALL cell lines. Despite this combination raising the levels of anti-apoptotic Bcl-2, it downregulated expression of c-Myc and hTERT, showing ZME as a potential adjuvant for treatment of pre-B-acute lymphoblastic leukemia [198]. Additionally, the telomerase inhibitor MST-312 decreased in vitro effective dose of doxorubicin. The combination MST-312/DOX reduced cell growth and promoted apoptosis in -B-ALL cells through unbalancing the Bax/Bcl-2 ratio aligned to down-regulation of c-Myc and hTERT [208]. It is important to mention that most TERT inhibitors are developed, aiming canonical function of telomerase, but there is evidence of the antitumor effect of MST-312 associated with non-canonical ones [207].
BIBR1532 is one of the most powerful telomerase inhibitors. This synthetic nonnucleoside compound binds to telomerase and acts as a chain terminator during nucleotide polymerization, inhibiting TA in a dose-dependent way [209,210]. BIBR1532 provoked cell death in pre-B-ALL cells after suppression of hTERT and c-Myc expression. Besides, high doses of BIBR1532 can induce p73, up-regulate Bax and activate caspase-3 [46]. Association of BIBR1532 with doxorubicin also reduced surviving expression and produced a synergistic anticancer effect in B-ALL through induction of ROS, which increased expression of Bax. Furthermore, it raised p21 levels, which promoted G1 cell cycle arrest and downregulation of p73-mediated c-Myc and hTERT expression [211].
To summarise, the combination of DOX with BIBR1532, ZME or MST-312 increases its therapeutic effect. A synergistic mechanism, if confirmed, could lead to therapeutical protocols with a lower dose of doxorubicin, thereby decreasing the risk of DOX-induced cardiotoxicity.
It is important to mention that there are a vast number of works showing the antitumor effects of telomerase inhibitors in a variety of in vitro and in vivo models of different cancer types [212], including some with which clinical trials are in progress or already concluded [213]. There are also studies testing telomerase-targeted immunotherapy [214] and other telomere-related therapeutical strategies. In this review, we attempted to summarize information regarding telomerase in B-ALL, which is still very scarce. Nevertheless, any one of these prototypes, once proved effective for cancer control, has the potential to be used in the context of leukemia.
In any case, further investigation is deeply required, including clinical trials, which could determine the safety of these compounds alone or as combined therapies for B-ALL patients. However, the greatest challenge that must be overcome in order to take studies with telomerase inhibition to the next level is the development of compounds targeting inhibition of non-canonical functions, which have been demonstrated to be crucial for cancer maintenance.

Conclusions
The physiological functions of telomerase in lymphocytes represent a challenge to determine the role of this enzyme in B-ALL. However, clearly reviewed data showed evidence of the potential of telomerase and other telomere-related proteins as clinical biomarkers and pharmacological targets. Briefly, high telomerase activity or hTERT expression, as well as short lymphocytes' telomeres, are frequently correlated with poor prognosis or even higher risk for B-ALL. However, there are still some apparent conflicting data in the literature, associating long telomeres with worse prognosis. We emphasize the word "apparent" since it also became clear that there is no single pattern concerning telomere and telomerase functions in leukemia, especially considering all possibilities of non-canonical actions of TERT. Additionally, the influence of telomerase on B-ALL seems to be divergent in different ethnic groups, which needs further investigation to be better elucidated. Finally, this pool of results shows a promising future for telomere and telomerase targeted therapy as new or combined treatments, but most data are too preliminary for short-term clinical use, especially in ALL.