Telomeres

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Telomeres, Telomerase and
Cancer
DR.KIRAN KUMAR BR

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TELOMERS
Telomeres are specialized nucleoprotein
complexes at the ends of linear chromosomes
consisting of long arrays of double stranded
TTAGGG repeats with G-rich 3’ singles.
Telomeres play a capping function by protecting the
ends of chromosomomes from degradation and
therby maintaining chromosomal integrity.

3. Structure of a Telomere

4. TELOMERASE
Telomerase is a specialized reverse transcriptase
that uses its RNA template to elongate the
telomeres by addition of 5’-TTAGGG-3’ repeats to
the terminal 3’ overhang.
It has two components- catalytic telomerase reverse
transcriptase(TERT) and telomerase RNA(TERC) .
Telomere elongation can be considered as the best-
known function of telomerase, and cell replication
capacity increases due to telomerase activity and
cell division sometimes continues indefinitely.

5. Gradual shortening of telomeres
-DNA replicates only in 5’-3’ direction and needs RNA
primer and complementary strands for this
replication.
-Hence after deletion of RNA primers on the lagging
strand, no other DNA can displace replicating the
region any longer.
- Initially it was taught that lagging strand was
responsible for telomere shortening and called this
problem the Lagging strand problem.

6. • However, considering the 3’ overhang structure
and inability of the leading strand in synthesizing
the 3’ overhang the leading strand problem was
finally introduced as the Chromosomal End
Replication Problem.

8. • Telomere shortening continues until replicative
senescence is triggered when the length of
telomeres is about 4–6 kb, also known as mortality
stage 1 (M1).
• Some cells manage to bypass M1 by inactivating
cell-cycle checkpoint pathways (e.g., p53 and or
p16/RB) and continue to shorten, eventually
entering mortality stage 2 (M2 or crisis)
• Characterized by genomic instability,
fusion/breakage mutagenic events, and massive
cell death.

9. • Very rarely, some cells can reactivate/upregulate
telomerase that is absent in most normal somatic
cells at M1 or M2 to stabilize telomere length,
leading to immortalization.
• Although immortalization is not sufficient to induce
malignant transformation, immortalization acquired
from activated telomerase in combination with
genome instability and mutation from telomere
shortening potentiates cancer formation.

10. Telomere Maintenance and Cancer
 Telomerase activity is observed in more than 80% of all
human cancers
 Human cancer cells are often significantly shorter than
their normal tissue counterparts.
 This suggests that telomere attrition has occurred
during the life history of these cancer cells, apparently at
very early phases of the transformation process when
telomerase activity is low
 The subsequent reactivation of telomerase restores
telomere function, but at a shorter set length.

11. Data from mouse models and correlative data
in staged human tumors, have indicated that a
lack of telomerase and associated telomere
attrition during the early stages of neoplastic
growth provides a potent mutator mechanism
.
This mechanism enables cancer cells to
achieve the high threshold of cancer-promoting
changes required to traverse the benign to
malignant transition.

12.  Reactivation of telomerase is critical to the
emergence of immortal human cells
 The preceding and transient period of telomere
shortening and dysfunction leads to carcinogenic
process through the generation of chromosomal
rearrangements
 Chromosomal rearrangements are brought about
through breakage-fusion-bridge (BFB) cycles

13.  A DSB(double strand break) created by BFB
cycles provides a nidus for amplification and/or
deletion at site of breakage for the resulting
daughter cells.
 The broken chromosome may become fused to
another chromosome, generating a second
dicentric chromosome and perpetuating the
BFB cycle.

15.  The accumulation of wholesale genetic changes
via aneuploidy, nonreciprocal translocations,
amplifications,and deletions (by the BFB cycles)
+ reactivation of telomerase gives rise to
carcinogenic changes & initiates
transformation process.

16.  In humans, the accumulation of oncogenic
lesions during normal aging or accelerated
accumulation of DNA damage (via
environmental carcinogen exposure or
oxidative damage) may:
-deactivate the telomere checkpoint response,
-accelerate telomere attrition,
- and drive the affected premalignant cells into
crisis.

17. Telomeric shortening can be viewed
-as a barrier to cancer development in the
presence of intact checkpoint response
-as a facilitator for the emergence of nascent
cancer cells in the absence of the
checkpoint response pathways.

19. Telomere-Induced Chromosomal
Instability
 Study of senescence and telomeres has
provided some insights into the link between
advancing age and increased cancer risk.
 In humans, there is a dramatic escalation in
cancer risk between the ages of 40 and 80,
primarily from a marked increase in epithelial
malignancies such as carcinomas of the breast,
lung, colon, and prostate.

20.  A conventional view is that the
cancer-prone phenotype of older
humans reflects the combined effects of
-cumulative mutational load,
-decreased DNA repair capabilities,
- increased epigenetic gene silencing, and
-altered hormonal and stromal milieus

21.  The measurement of telomerase activity in
adenomatous polyps and colorectal cancers
-Telomerase activity is low or undetectable in small and
intermediate-sized polyps, reflecting less intact
telomere function.
-Telomerase activity increases markedly in large
adenomas and colorectal carcinomas, reflecting
stabilization of telomere function.
-Proves that widespread and severe chromosomal
instability is present early on during human tumorigenesis
at a time when telomerase activity is low.

22. Telomere Dynamics, Inflammatory Diseases,
and Cancer:
High cancer incidence is associated with diseases
characterized by-chronic cell destruction and renewal
as well as inflammation.
-Example is the high incidence of hepatocellular
carcinoma in late-stage cirrhotic livers.
-Cirrhotic livers show a documented reduction in
telomere length over time.
-congenital telomerase deficiency may be predisposed to
fibrotic liver disease, including cirrhosis.

23. -Another example of a telomere-based pathogenic
relationship between chronic tissue turnover, telomere-
based crisis, and increased cancer risk is ulcerative
colitis
-In addition to the progressive telomere attrition resulting
from the cell turnover, accelerated telomere attrition
might occur via increased oxidative stress and from the
altered inflammatory microenvironment.

24.  Serial analyses of telomere length from these
tissues may provide prognostic information
regarding the rising risk of cancer development.
The progress in understanding of telomere
biology has mechanistically connected diverse
fields in medicine involving
 chronic inflammatory diseases,
 degenerative diseases,
 geriatrics, and
 oncology.

25. Telomerase and Telomere Maintenance As
Therapeutic Targets
-Cell culture–based studies of human cancer cells have
established that inhibition of telomerase culminates in cell
death after extended cell divisions.
-Intense efforts to design therapeutic strategies capable of
targeting telomere structure and the telomerase
holoenzyme function.

26. • High telomerase expression and activity in cancer
cells, compared to low levels of telomerase
observed in very few adult stem cell compartments,
such as hematopoietic, epidermal, and
gastrointestinal cells, have fueled significant efforts
to target telomerase therapeutically.
• Several strategies are currently in various stages of
development and are discussed based on
mechanism of action.

27. (1) Telomere-based strategies
(2) Telomerase interference
(3) Direct telomerase inhibition
(4) TERT or TERC promoter driven therapy
(5) Telomerase immunotherapy.

28. Telomere-Based Strategies
• Telomere-based approaches offer a potential
advantage in that they directly mimic or interfere
with telomere structures rather than disrupting
telomerase.
• Hence, these strategies may be effective even for
telomerase-negative malignancies (e.g., sarcomas,
some lymphomas) that maintain their telomeres
using alternative lengthening of telomeres

29. • G-quadruplex stabilizers: G-quadruplex is a
tetrad planar structure formed by four guanine
bases stabilized by Hoogsteen hydrogen bonds,
and this structure can be formed in guanine rich
nucleic acids including telomeres.
• They prevent unwinding of the quadruplex, making
the 3’ overhang inaccessible to telomerase and
thus prevents telomere lengthening by telomerase
or by ALT . This accelerates telomere shortening
and subsequent cell death .

30. - Several G-quadruplex stabilizers, including
TMPyP4(Tosylate), RHPS4 and telomestatin,
have been tested and induced in vitro cell growth
arrest, increased apoptosis, in vivo tumor xenograft
shrinkage.
- These are still in early development and have not
entered clinical trials.
- Quarfloxin/CX-3543 has entered phase I and II
trials but is thought to induce apoptosis through
inhibition of ribosomal RNA (rRNA) biogenesis.

31. • T-oligo: T-oligo is an 11-mer oligonucleotide that is
homologous to the 3’-telomeric overhang.
• Introduction of T-oligo into cancer cells mimics the
presence of uncapped telomeres and induces a
DNA damage response (DDR), apoptosis, and
autophagy.
• A study performed in melanoma cells showed that
T-oligo increased p53 activity, resulting in cellular
differentiation and apoptosis.

32. • Tankyrase inhibitors: Tankyrase belongs to the
poly (ADP-ribose) polymerase (PARP) protein
superfamily that is involved in various cellular
processes, including telomere length regulation .
• Telomere lengthening requires dissociation of
TRF1 from the telomere in order that telomerase
gain access to the telomere. Tankyrase facilitates
TRF1 dissociation from the telomere via poly(ADP-
ribosyl)action of TRF1, followed by ubiquitin-
mediated degradation of TRF1.

33. • Therefore, tankyrase inhibition reduces
dissociation of TRF1 from the telomere and
prevents binding of telomerase to telomere, thus
inhibiting telomere lengthening.
• Several tankyrase inhibitors (e.g., IWR1, IWR2,
JW55, flavone, and XAV939) are being tested but
have not as yet entered clinical trials.

34. • Direct Telomerase Inhibition
• GRN163L (Imetelstat), was developed by Geron, as a novel
anticancer agent.
• Imetelstat is a 13-mer oligonucleotideN3’-P5’ that is
complementary to nine nucleotides in the template region and
four nucleotides 5’ of the template region of TERC.
• By directly binding the TERC template, Imetelstat disrupts
telomerase ribonucleoprotein (TERT + TERC) assembly and
enzymatic activity at telomeres.
• In vitro, this results in telomere shortening and eventual DNA
damage and cell death.

35. Telomerase Interference
• Altered TERC templates introduce by lentiviral
infection into cancer cells induced foci of DNA
damage at telomeres and characteristic “anaphase
bridges” caused by telomeric fusions.
• Ultimately led to cancer cell apoptosis and
decreased proliferation in vitro and in xenograft
models

36. TERT or TERC Promoter Driven Therapy
• Increased TERT promoter activity and TERT expression is a
hallmark of most cancer types.
• Recently a highly recurrent TERT promoter mutation
(C250T) was described that creates a unique site for the
binding of protein complexes containing E-twenty-six (ETS)
and p52 subunit for enhanced TERT expression, increased
telomerase activity and subsequent tumorogenesis .
• Correction of this mutation and reduction of TERT expression
may ultimately be achievable using recently developed gene
editing techniques.

37. • Oncolytic virus: To explore a telomerase-specific oncolytic
therapy, a type 5 adenovirus was constructed by inserting
adenovirus E1A and E1B genes under the control of the
human TERT promoter.
• Upon infection, E1A and E1B are expressed and induce viral
replication and cellular lysis in TERT promoter active tumor
cells, while sparing TERT promoter inactive benign cells.
• OBP-301 (telomelysin) has been shown to selectively lyse
lung cancer cells , kill CD133+ human gastric cancer stem-
like cells, and inhibit lung tumor xenografts treated with direct
intratumoral injection.
• Currently, OBP-301 is in a phase1/2 study in patients with
hepatocellular carcinoma.

38. TELOMERASE IMMUNOTHERAPY
-Immunotherapy, targeting immune recognition and the destruction of
cells that express telomerase.
 Immune responses, specifically cytotoxic T-cell responses, have
been generated against peptide sequences of the hTERT protein,
 These cytotoxic T cells are capable of selectively lysing target cells
that express TERT peptides presented on the cell surface in the
context of MHC class I molecules.

39. • Telomerase based immunotherapy can be divided
into two approaches:
(1)direct immune activation in vivo
(2) ex vivo activation and expansion of immune cells.
. Direct activation using TERT derived peptide-GV1001: GV1001
consists of 16 amino acids and is recognized by both MHC
class I and class II molecules, thus offering the advantage of
eliciting both CD8+ and CD4+ responses.

40. • Several clinical trials have been conducted in non-
small cell lung cancer (NSCLC), pancreatic cancer,
hepatocellular carcinoma, and malignant
melanoma.
• No serious adverse events were observed in
patients treated with GV1001, with mostly grade 1
or 2 injection site reaction.

41. • Ex vivo activation and expansion of immune
cells using GRNVAC1:
• In this autologous vaccination approach, patient-derived
dendritic cells are isolated and transfected ex vivo with mRNA
encoding a chimeric protein, then administered to patients
through intradermal injections.
• This approach triggered TERT-specific CD4+ and CD8+ T
cell responses, and treatment was well tolerated with only
grade 1 toxicities.

43.  Data from the telomerase-deficient mouse model has
also shown that cells and animals with telomere
dysfunction are
more sensitive to ionizing radiation and
chemotherapeutic agents.
 The combination of increased DNA damage with reduced
capacity for normal repair may also produce marked
increases in the toxicity of chemoradiotherapy.

44. Costs of Senescence
 Activation of senescence pathways depletes
tissue-specific stem cell compartments, leading to a
decrease in tissue regenerative capacity and aging.
 Relevant in clinical oncology as many of the
cytotoxic agents such as chemo- and radiotherapy
intended to treat cancers untowardly promote
senescence, and may hasten aging.

45. Therefore, senescence may be a cause of many of
the long-term toxicities of cancer therapy; for
example ,
 reduced bone marrow reserve after alkylating
agents exposure,
 poor wound healing in fields of prior radiotherapy,
and
 radiation-induced fibrosis

46. Telomeres and Aging
 Telomere length declines with aging in humans
 Telomere dysfunction and telomere-induced
senescence might increase with aging.
 Several studies have explored the use of telomere
length determination in accessible peripheral blood
lymphocytes rather than the diseased tissue.
 Peripheral blood lymphocyte telomere lengths can
provide predictive information on the risk of
developing atherosclerosis, premature myocardial
infarctions, coronary artery disease, Alzheimer's
disease status, and overall mortality.

47.  More direct proof of a role of telomere dysfunction
in a few forms of age-induced organ failure has
been suggested by the identification of germline
TERC and TERT mutations associated with
unusually short telomeres.
 Such patients appear to be at increased risk for
forms of bone marrow failure (aplastic anemia and
myelodysplasia) and idiopathic pulmonary fibrosis.

48.  Clinical Relevance
-Measurement of the expression of senescence
markers could in turn predict a tissue's future
regenerative capacity.
- For example, increased expression of senescence
markers in the bone marrow might forecast
increased myelotoxicity from chemotherapy.
-Additionally, agents that inhibit the induction of
senescence in response to DNA damaging agents
might also spare long-term chemotherapy toxicity

49. The pivotal role of telomere attrition in the
pathogenesis of cancer and tissue aging provides
potential avenues for
 development of cancer risk biomarkers,
 diagnostics, and
 rationally designed therapeutics.

50.  THANK YOU !!!!