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Aging and the Telomere Connection

Dr. Jerry W. Shay


Distinguished Chair in Geriatrics Research
UT Southwestern Medical Center (Dallas, Texas)

Everyone Recognizes Aging

Disclosure
Jerry W. Shay
The following potential conflict of interest relationships are germane to my
presentation.
Employment: None
Speakers Bureau: None
Stock Shareholder: None
Grant/Research Support: None
Scientific Advisor: Life Length

Status of FDA devices used for the material being presented


NA/Non-Clinical
Status of off-label use of devices, drugs or other materials that constitute
the subject of this presentation
NA/Non-Clinical

Promising Technologies for Aging Research

Neuroendocrine Interventions
AGE Breakers (Advanced Glycation End Products)
Anti-inflammatory Agents
Antioxidant Supplements
Caloric Restriction Mimetics
Mitochondrial Modulators
Stem Cell Therapies
Telomere Extension - Cell Rejuvenation

Promising Technologies for Aging Research

Neuroendocrine Interventions
AGE Breakers (Advanced Glycation End Products)
Anti-inflammatory Agents
Antioxidant Supplements
Caloric Restriction Mimetics
Mitochondrial Modulators
Stem Cell Therapies
Telomere Extension - Cell Rejuvenation

What is Aging?

Aging is associated with the gradual decline in


performance and reserve capacity in organ
systems
Aging of organ systems is often associated with a
decrease in the number and/or function of cells
Old cells do not maintain and repair tissues as
well as young cells, leading to decreased overall
vitality

1930 - 1940: Beginning to Understand the End

Hermann J. Mller

Barbara McClintock

X-ray mutagenesis
experiments in Drosophila

Cytogenetics of maize mutants

Absence of terminal
deletions or inversions after
irradiation

Chromosomes lacking telomeres


form end-to-end fusions

Telomeres: special functional complexes at the


end of eukaryotic chromosomes

Chromosomes are Capped by Telomeres


telo (end) mere (segment)

Why Should You Care About Telomeres?

Telomere Hypothesis of Aging

Telomeres: TTAGGG repeats at the ends of linear


chromosomes are progressively lost with each
cell division
End-replication problem, oxidative damage,
and other end processing events

Senescence (aging) occurs when a few


telomeres are short

Telomeres of a young
or quiescent cell

Stop

Telomeres of a senescent (aging) cell


or a cell with uncapped ends

In Vitro: Replicative Senescence in Normal Cells


STOP

Hayflick, 1950s
STOP

STOP

STOP

STOP

STOP

STOP

STOP

Short telomeres correlate with


replicative senescence

Harley et al,

In Vivo: Telomeres Shorten With Increased Age


Tissue Source

Telomere Length (kb)

Sperm
Placenta

Fetal brain
Fetal kidney
Colon mucosa (30-65 yrs)
Colon mucosa (65-88 yrs)
Blood (20-39 yrs)
Blood (40-59 yrs)
Blood (60-79 yrs)

N. Hastie .R. Allshire Nature, 346: 866

12

16

Short telomeres correlate with increased age

20 (kb)

Adverse Consequences of Short Telomeres

Loss of tissue renewal capacity

Failure of stem cell niches

Induction of metabolic dysfunction

Senescence-associated secretory phenotype (SASP)

Increased risk of cancer (genomic instability)

Cancer Incidence

Aging and Cancer:


A Double-edged Sword

Age
Cancer Rises with Increased Age

Replicative Senescence May Be a Potent


Anti-Cancer Protection Mechanism
Senescence
brick wall
1

Cancer

106
cells

106
cells

106
cells

106
cells

106
cells

106
cells

106
cells

106
cells
cell doublings

Short Telomeres are Present in Almost All


Preneoplastic Lesions

Ductal Carcinoma In Situ (DCIS)


breast cancer
Prostatic Intraepithelial Neoplasia (PIN)
prostate cancer
Cervical Intraepithelial Neoplasia (CIN)
cervical cancer
Barretts esophagus
esophageal cancer
Ulcerative colitis
colorectal cancer
Liver cirrhosis
hepatocellular carcinoma
Myeloproliferative disorders
decreased general immunity, leukemia

Telomere Hypothesis of Cancer

Most pre-malignant tissues have very short telomeres


Telomere attrition may initially be a tumor
suppressor pathway but in combination with other
cellular alterations may drive genomic instability
Cancer cells are almost always immortal
Must engage a mechanism for stabilizing telomere
length for the growth of the advanced tumor

Hallmarks of Cancer
Hanahan and Weinberg

Cell 2011

Telomerase is Detected in ~90% Human Cancers

Telomere
hTR
template

hTERT
TRAP

Telomerase is a molecular motor that adds


new DNA onto the ends of telomeres
hTERT = human telomerase reverse transcriptase

ITAS

Normal

Cancer

(expressed in cancer cells and some normal stem cells)


hTERT

hTR/hTERC = human telomerase template RNA


(constituitively expressed but up regulated in cancer)

GAPDH

Lasker Award for Basic Science 2006


Nobel Prize in Medicine 2009

Elizabeth Blackburn

Jack Szostak

Carol Greider

For the prediction and discovery of telomerase, a


remarkable RNA-containing enzyme that synthesizes the
ends of chromosomes, protecting them and maintaining
the integrity of the genome.
Laskerfoundation.org September 17, 2006

Individuals With Malignant Tumors Without


Telomerase Activity Have Better Outcomes
IVS

Survival
rate (%)

NT

100

Low or Nil Telomerase Activity


(n = 82)

Neuroblastoma IVS
9.4
6.6

50

High Telomerase Activity


(n = 23)

4.4
0

10

(year)

2.3
(Kb)

TRF

Suggests inhibition of telomerase may be a


potent anti-cancer approach

Approaches to Inhibiting Telomerase For Cancer


Therapy

Imetelstat: Direct Telomerase Enzyme Inhibitor


GRN163L: 3 NH2-AACAGATTGGGAT-OH 5'
hTERC:
5UUGUCUAACCCUAAC3'

hTERC or hTR
RNA Template
Telomerase

A U C C C A A U C UGUU
T AGGG T T AGA C A A

T T AGGG T T

Telomere

GRN163L
Imetelstat

GRN163L (Imetelstat)
13-mer lipidated thio-phosphoramidate
backbone complementary to the
telomerase RNA template with a long
half-life in human tumors

Potent competitive telomerase template


antagonist (oligonucleotide)
(not antisense that targets mRNA)
Mechanism of action: competitive with
telomere binding

Inhibits telomerase and telomeres


shorten

0
H 1703
H 661
H 1299
H 1975
H C C 4006
H 1993
H 1568
H C C 1359
H 1819
H 2882
H 2009
H 1693
H 226
H 838
H 2126
H 2347
H 1355
H O P 62
H 460
H 1155
H 157
H C C 2429
A5 4 9
H 322
H 1395
H 596
H 1792
H 3122
H 1666
H 2122
H 727
H 650
H C C 78
H 2228
H 358
H 2291
H 3255
H 1373
H 441
H C C 1438
H C C 44
H 2073
H 2087
H C C 95
H C C 827
H 522
H 1437
H 1838
H C C 4019
H 23
H C C 2279
H 1650
H 920
H C C 193
H C C 1833
C a lu - 6
H 1648
C a lu - 1
H 1944
H C C 515
H 820
H 2887
C a lu - 3

T e lo m e r e L e n g t h ( k b )
25

20
T o ta l P o p u la tio n D o u b lin g s

15

(Robin Frink, John Minna)


H 460

100

H 460
1 u M Im e te ls ta t

50

0
0
50
100

D ays

T o ta l P o p u la tio n D o u b lin g s

Most Cancer Cells Have Short Telomeres: Response


Time to Imetelstat is Faster in Cells with Short Telomere
Length
150

150
200
250
60

C a lu -3

40

C a lu -3
1 u M Im e te ls ta t

20

0
0
20
40

D ays
60

10

5
80
100

Imetelstat (GRN163L): Phase II Trials

Non Small Cell Lung Cancer Randomized Phase II


study of Imetelstat maintenance after first line
(debulking) induction chemotherapy, +/- Bevacizumab,
(Bev) primary end point, PFS, ~116 patients enrolled,
interim analysis trend in PFS in favor of the Imetelstat
arm.
Arm

Imetelstat

No Imetelstat

Total

Bev

27

13

40

No Bev

50

26

76

Total

77

39

116

Analysis of Phase II (CP14B-012) Imetelstat


NSCLC Trial Overall Survival
1
Survival Results (N=114)
0.9

Overall Survival Rate

0.8

Imetelstat

Control

14.3

11.5

Median
(95% CI)

0.7
0.6
0.5
0.4
0.3
0.2

Control

0.1

Imetelstat

0
0

10

12

14

16

18

20

22

24

Months Since Randomization


N at risk

114 104 91 84 69 50 41 25 18 11

Retrospective Analysis of Phase II (CP14B-012) Imetelstat


NSCLC Trial Telomere Length and Progression Free and
Overall Survival
Short Telomeres
1

Control
Imetelstat

0.9

Control
Imetelstat

0.8

0.9

0.7

0.8

Overall Survival Rate

Progression-free Survival Rate

Short Telomeres

0.6

0.5
0.4
0.3
0.2
0.1

0.7
0.6
0.5
0.4
0.3
0.2

0.1
0

0
0

10

12

14

16

Months Since Randomization

18

20

10 12 14 16 18 20

Months Since Randomization

Cancer Patients with Short Telomeres Respond Better to Telomerase Inhibitors

Ongoing and Future Studies with Imetelstat

Lung cancer patients with short telomeres have rapid


progression of disease
While not statistically significant patients on the
Imetelstat arms, showed an overall survival trend
Suggests future trials should target subsets of cancer
patients with the shortest telomeres (e.g. short
telomeres may be an enrichment biomarker).

Two Faces of Telomerase:


Dr. Jekyll and Mr. Hyde

Cancer-permissive

Aging cell rejuvenation

Telomerase Extends Cellular Lifespan


(Cellular Fountain of Youth)
Expression of hTERT in mortal cells is sufficient to
generate telomerase activity, lengthen telomeres,
and extend cellular lifespan
BJ
-

PD

Population Doublings

hTERT

30 kb

7.7 kb

ITAS

160
140

hTERT +

120
100
80

hTERT -

60
40
0

50

100

150

200

250

300

Days
TRAP

TRF

Telomeric repeat
amplification protocol

Terminal restriction fragments

ITAS = internal telomerase amplification standard


PD = population doubling

Immortalization of Normal Cells with hTERT Does


Not By Itself Transform Cells
Normal

Cancer

hTERT +

Contact inhibition of growth

present

absent

present

Growth factor requirements

high

low

high

present

absent

present

intact

absent

intact

Tumors in nude mice

normal
absent

abnormal
present

normal
absent

Proliferative life span

finite

indefinite

indefinite

Characteristics

Anchorage dependence
Cell cycle checkpoints

Karyotypic profile

Evidence for Telomere Shortening in Human


Genetic and Chronic Diseases

Chronic wounds

Myeloproliferative disorders

Macular degeneration

Arteriosclerosis

pressure ulcers, diabetic ulcers

decreased general immunity

retinal epithelium, endothelium


vascular endothelium
angiogenesis responses

Dyskeratosis congenita

dyskerin or hTR mutations


Idiopathic pulmonary fibrosis
hTERT or hTR mutations
Muscular dystrophy
dystrophin mutation
Liver cirrhosis
hepatocellular carcinoma
Barretts esophagus
esophageal cancer
Ulcerative colitis
colorectal cancer

Mutations in Telomerase are Genetic Risk Factors


for Clinical Disease: Telomeropathies

Peripheral Blood Mononuclear Cell Telomeres are


Shorter in Patients With Idiopathic Pulmonary Fibrosis

Normal
Mutation without IPF
Mutation with IPF
Tsakiri et al, 2007

Peripheral Blood Lymphocyte Telomeres are Shorter in


Patients With Dyskeratosis Congenita

Lansdorp lab Blood, 2007

Mutations in Components of the Telomerase Complex


May Lead to Premature Stem Cell Depletion
DKC/IPF
TERT/TERC
Mutations

Telomere
Shortening

Cell cycle arrest


(senescence)
Progenitor stem cell depletion

Haploinsufficiency in telomerase leads to


premature stem cell depletion and clinical
disease.
Telomerase is not in excess.

Clinical
Disease

Telomere length

Telomere Length: Biomarker of Cellular Aging

Health

Disease risk
30-40

Age (years)

70-80

Influence of Telomeres on Cardiovascular Health

Reduced telomere lengths are found in patients with


cardiovascular risk factors such as atherosclerosis,
hypertension, obesity, diabetes, smoking, physical
inactivity, stress, and chronic infections.
Shorter telomeres have been associated with increased
incidence of diseases and poor survival.
A positive effect on telomere length is found with increased
physical activity, statins for treatment of high cholesterol
and higher blood levels of omega-3 fatty acids.

Telomere length

Stress, Diet, Inflammation Leads to Oxidative DNA


Damage and Can Accelerate Telomere Shortening

Health

Disease risk
50-60 70-80

Age (years)

Diet and Nutrition Can Modify Telomere Length


Omega 3
polyphenols
curcumin

Vitamins
C&E

Vitamins
A&D
Oxidative stress

DNA damage
Inflammation

Telomere
shortening

Telomere Length

Potential of Telomerase Activation: Are Telomeres


Targets of Age-Associated Disease?

Transient telomerase activation or natural


products that activate telomerase

Cancer cell

Cell Divisions

TA-65 is a Single Molecule Isolated from Astralagous


Astralagous as been prescribed for
centuries for general weakness,
chronic illnesses, and to increase
overall vitality
1. Hepatoprotective properties

2. Anti-inflammatory properties
3. Antioxidant properties
4. Immune stimulating properties
5. Antiviral properties

6. Antibacterial properties
7. Cardiovascular effects

Astragalus membranaceus

Possible Effects of Transient Telomerase Activation


in Cells of Patients

Slow the rate of telomere loss


Improved immune cell structure/function
Prevention or slow down rate of genomic instability?
Activation of renewal pathways
Increase repair, resistance to stress-induced apoptosis
Increased doublings for normal cells = increased chance
of mutations occurring
Increased doublings for premalignant cells = increased
chance of mutation to next tumor stage

Telomere length

Will Telomere Length Modification Delay Cellular


Aging in Healthy Individuals?

Health

Disease risk
70-80 90-100

Age (years)

Science 232: 414-415 (April 22, 2011)

What Do You Need to Know if You Want to


Determine Your Biological Not Chronological Age

Why do you need to know your biological age?


If your biological age is greater than your chronological
age what does this mean and what can be done to slow
down or reverse this?
What is the difference between average telomere
lengths and short telomeres and why is this important?
What is the standard/normal telomere length by age?
What is the best and most accurate telomere test?
What cell types should you test?

Methods for Measuring Telomere Length


Scalability
Blasco

Lansdorp
Flow cytometry
Multiple
sample
processing

Blackburn
Harley

HT qFISH (blood)

qPCR
Southern Blot
TRF analysis
Dot blot
Blasco

Single
sample
processing

STELA
qFISH Telomapping (tissue)

Average telomere length

Percentage of short telomeres

Methods for Measuring Telomere Length


Scalability
Blasco

Lansdorp
Flow cytometry
Multiple
sample
processing

Blackburn
Harley

HT qFISH (blood)

qPCR
Southern Blot
TRF analysis
Dot blot
Blasco

Single
sample
processing

STELA
qFISH Telomapping (tissue)

Average telomere length

Percentage of short telomeres

2009 Nobel Prize Winner for the Discovery of


Telomerase Dr. Carol Greider

Fluorescence In Situ Hybridization (FISH) for


Measuring Telomeres
FISH provides a sensitive method for specific telomere length detection

Q-FISH or quantitative fluorescence in situ hybridization

DAPI signal in blue (chromosomes)


CY3 signal in yellow (telomeres)

short telomeres

long telomeres

HT Q-FISH Telomere Length Measurements


1) Blood sample

2) HT Q-FISH

3) Confocal microscopy

About one teaspoon

6) Data analysis

5) Data processing

4) Capture of individual
telomere signals

Mean TL
60

frequency

% Short telomeres
40

20

0
0

10

15

20

mean telomere length (kb)

Canela & Vera et al., PNAS, 2007

20
15
10
5
0
0

10

20

30

40

Age (years)

50

60

70

% short telomeres (<3kb)

Mean telomere length (Kb)

The Percent of Short Telomeres, Not Average


Telomere Length Determines the Onset of Disease
60
50
40
30
20
10
0
0

10

20

30

40

50

60

70

Age (years)

Percent of short telomeres detect more differences between individuals than


average length
Percent of short telomeres show higher scattering as we age
Percent of short telomeres are likely to reflect both genetic and lifestyle
choices (environment)

Telomapping (Tissue Telomere FISH)

Normal Prostate
Telomere FISH

Basal
cells

Prostatic
Intraepithelial
Neoplasia (PIN)

Luminal epithelial cells

Stromal fibroblast cells


Alan Meeker

Determining average telomere length analysis on this biopsy would not be useful

Its the shortest telomeres that lead to age-associated diseases

Issues to Consider When Deciding on a Test for


Telomere Length

Cell type to analyze: Saliva versus blood


Saliva is easy to obtain and testing can be less
expensive
However, saliva contains few living cells (statistical
problems); poor quality of DNA (significant
degradation and contaminated with bacteria); no
published data comparing results to other tissues in
the body; only average telomere length provided
Blood a bit more work to obtain; testing can be more
expensive
However, blood contains many living cells, high
quality DNA; robust literature on telomere biology and
correlation with other tissues; average and shortest
telomeres can be provided.

Issues to Consider When Deciding on a Test for


Telomere Length

Reliability Is the test accurate within 5-10% if one


conducted 3 separate preparations from the same
individual?
Does the test provide both average and percent of
shortest telomeres? Average telomere length may be
less useful since a single critically short telomere may be
sufficient to initiate cell senescence (and disease onset).

Is there a detailed questionnaire about lifestyle and


health issues including family history, so longitudinal
studies are more meaningful?

Why Use Telomere Length Determinations in Your


Practice?

To identify inherited telomere disorders in patients and


families (telomere-induced disease states)
To identify healthy individuals with accelerated
biological aging
To enable early intervention-lifestyle modifications to
reduce stress, inflammation, oxidative damage and other
inducers of accelerated telomere loss
To provide therapeutic interventions to slow down or
reverse telomere loss (stem cells, bone marrow
transplantations to select optimal donors, tissue
engineering, supplements)

Telomeres and Aging Recent Headlines

Science Daily, March 27, 2013. Short telomeres are not only a
marker for higher risk of disease and mortality, but are a likely
underlying cause of several age-related diseasesincluding heart
disease and various cancers. The team measured telomere lengths
in over 48,000 individuals and identified seven genetic variants that
are associated with telomere length, and found that these genetic
variants also affected risk of various diseases.
EurekAlert, March 9, 2013. Scientists at the Intermountain Heart
Institute Salt Lake City, showed that people with longer telomeres
live longer. The study's lead researcher, Dr. John Carlquist, said of
telomere testing "We can already test cholesterol and blood
pressure of a patient to see how treatment is working, but telomere
testing could give us a deeper view into how the treatment is
affecting the body and whether or not the treatment is working.

Telomeres and Aging Headlines

Time Magazine, February 22, 2013. Scientists at Carnegie Mellon


University report that short telomere lengths correlate with susceptibility
to common cold infections
Genetic Engineering & Biotechnology News, January 24, 2013.
Research from the Spanish National Cancer Research Centre study
shows caloric-restriction slows the rate of telomere shortening
The New York Times, January 27, 2010. German researchers find that
middle-age people with more consistently active lifestyles had
telomeres on average 40% longer than those who were primarily
sedentary.
The Globe and Mail, January 20, 2010. Researchers from the University
of California, San Francisco, report that patients with coronary artery
disease who had the highest blood levels of omega-3 fats had a slower
rate of telomere shortening than those with less omega-3.

Conclusions

The analysis of human telomere length has become a


central validated biomarker that reflects biological (not
chronological) human aging processes.
Accurate commercial tests are now available that will
lead to a better understanding of the relationship of
telomeres and telomerase to aging and cancer.
Products currently being touted to promote healthy aging
may wish to consider incorporating telomere length
testing to determine if telomere biology may be the
mechanism responsible.

Youth is a gift of nature, but age is a work of art


G. Kanin

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