aging and the telomere connection-dr.jerry shay-a4m-orlando-april 2013

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

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. Müller

X-ray mutagenesis

experiments in Drosophila

Absence of terminal

deletions or inversions after

irradiation

Barbara McClintock

Cytogenetics of maize mutants

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

Short telomeres correlate with

replicative senescence

STOP

STOP

STOP

STOP

STOP

STOP

STOP

Hayflick, 1950s

Harley et al,

STOP

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)

0 12 4 16 8 20 N. Hastie ….R. Allshire Nature, 346: 866 (kb)

Short telomeres correlate with increased age

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)

Aging and Cancer:

A Double-edged Sword

Age

Can

cer

Incid

en

ce

Cancer Rises with Increased Age

Replicative Senescence May Be a Potent

Anti-Cancer Protection Mechanism

106

cells

106

cells

106

cells

106

cells

Cancer

cell doublings

1 2 3 4 5 7 6

106

cells

106

cells

106

cells

8

106

cells

Senescence

“brick wall”

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

• Barrett’s 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

Telomerase is a molecular motor that adds

new DNA onto the ends of telomeres

hTERT = human telomerase reverse transcriptase (expressed in cancer cells and some normal stem cells)

hTR/hTERC = human telomerase template RNA (constituitively expressed but up regulated in cancer)

hTERT hTR

template

Telomere

TRAP

ITAS

Normal Cancer

GAPDH

hTERT

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

5 10 (year)

Survival rate (%)

100

50

0

High Telomerase Activity

Low or Nil Telomerase Activity

( n = 82)

( n = 23)

Suggests inhibition of telomerase may be a

potent anti-cancer approach

Neuroblastoma IVS

TRF

N T

IVS

2.3

4.4

6.6

9.4

(Kb)

Approaches to Inhibiting Telomerase For Cancer

Therapy

Imetelstat®: Direct Telomerase Enzyme Inhibitor

• Potent competitive telomerase template

antagonist (oligonucleotide)

• (not antisense that targets mRNA)

• Mechanism of action: competitive with

telomere binding

• Inhibits telomerase and telomeres

shorten

13-mer lipidated thio-phosphoramidate

backbone complementary to the

telomerase RNA template with a long

half-life in human tumors

GRN163L (Imetelstat)

GRN163L: 3 ‘NH2-AACAGATTGGGAT-OH 5'

hTERC: 5‘…UUGUCUAACCCUAAC…3'

A U C C C A A U C U G U U

Telomerase

T A G G G T T A G A C A A

A G G G T T GRN163L Imetelstat

hTERC or hTR RNA Template

T T

Telomere

Most Cancer Cells Have Short Telomeres: Response

Time to Imetelstat® is Faster in Cells with Short Telomere

Length

H1

70

3H

66

1H

12

99

H1

97

5H

CC

40

06

H1

99

3H

15

68

HC

C1

35

9H

18

19

H2

88

2H

20

09

H1

69

3H

22

6H

83

8H

21

26

H2

34

7H

13

55

HO

P6

2H

46

0H

11

55

H1

57

HC

C2

42

9A

54

9H

32

2H

13

95

H5

96

H1

79

2H

31

22

H1

66

6H

21

22

H7

27

H6

50

HC

C7

8H

22

28

H3

58

H2

29

1H

32

55

H1

37

3H

44

1H

CC

14

38

HC

C4

4H

20

73

H2

08

7H

CC

95

HC

C8

27

H5

22

H1

43

7H

18

38

HC

C4

01

9H

23

HC

C2

27

9H

16

50

H9

20

HC

C1

93

HC

C1

83

3C

alu

-6H

16

48

Ca

lu-1

H1

94

4H

CC

51

5H

82

0H

28

87

Ca

lu-3

0

5

1 0

1 5

2 0

2 5

Te

lom

ere

Le

ng

th (

kb

)

(Robin Frink, John Minna)

0 2 0 4 0 6 0 8 0 1 0 0

0

2 0

4 0

6 0

C a lu -3

C a lu -3

1 u M Im e te ls ta t

D a y s

To

tal

Po

pu

lati

on

D

ou

bli

ng

s

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0

0

5 0

1 0 0

1 5 0

H 4 6 0

H 4 6 0

1 u M Im e te ls ta t

D a y s

To

tal

Po

pu

lati

on

D

ou

bli

ng

s

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

0 2 4 6 8 10 12 14 16 18 20 22 24

Months Since Randomization

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1 O

vera

ll Su

rviv

al R

ate

N at risk 114 104 91 84 69 50 41 25 18 11 5 1 1

Control Imetelstat

Survival Results (N=114)

Imetelstat Control

Median

(95% CI) 14.3 11.5

Retrospective Analysis of Phase II (CP14B-012) Imetelstat®

NSCLC Trial – Telomere Length and Progression Free and

Overall Survival

Short Telomeres

0 2 4 6 8 10 12 14 16 18 20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Pro

gre

ssio

n-f

ree

Surv

ival

Rat

e

Control

Imetelstat

0 2 4 6 8 10 12 14 16 18 20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Ove

rall

Surv

ival

Rat

e

Control

Imetelstat

Short Telomeres

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

hTERT - +

ITAS

TRAP

7.7 kb

30 kb

PD

TRF

0 50 100 150 200 250 300

40

60

80

100

120

140

160

Po

pu

lati

on

Do

ub

lin

gs

Days

hTERT +

hTERT -

Telomeric repeat

amplification protocol Terminal restriction fragments ITAS = internal telomerase amplification standard

PD = population doubling

Characteristics

Contact inhibition of growth

Growth factor requirements

Anchorage dependence

Cell cycle checkpoints

Karyotypic profile

Normal

present

high

present

intact

normal

Cancer

absent

low

absent

absent

abnormal

hTERT +

present

high

present

intact

normal

Immortalization of Normal Cells with hTERT Does

Not By Itself Transform Cells

Characteristics

Proliferative life span

Normal

finite

Cancer

indefinite

hTERT +

indefinite

Tumors in nude mice absent present absent

Evidence for Telomere Shortening in Human

Genetic and Chronic Diseases

• Chronic wounds

– pressure ulcers, diabetic ulcers

• Myeloproliferative disorders

– decreased general immunity

• Macular degeneration

– retinal epithelium, endothelium

• Arteriosclerosis

– 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

• Barrett’s 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

Clinical

Disease

Haploinsufficiency in telomerase leads to

premature stem cell depletion and clinical

disease.

Telomerase is not in excess.

Telomere Length: Biomarker of Cellular Aging

Health

Disease risk

Age (years)

Telo

me

re le

ng

th

30-40 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.

Stress, Diet, Inflammation Leads to Oxidative DNA

Damage and Can Accelerate Telomere Shortening

Health

Disease risk

Age (years)

Telo

me

re le

ng

th

50-60 70-80

Diet and Nutrition Can Modify Telomere Length

Oxidative stress

Inflammation

DNA damage

Telomere

shortening

Vitamins

C & E

Vitamins

A & D

Omega 3

polyphenols

curcumin

Potential of Telomerase Activation: Are Telomeres

Targets of Age-Associated Disease?

Cell Divisions

Telo

mere

Len

gth

Transient telomerase activation or natural

products that activate telomerase

Cancer cell

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

TA-65 is a Single Molecule Isolated from Astralagous

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

Will Telomere Length Modification Delay Cellular

Aging in Healthy Individuals?

Health

Disease risk

Age (years)

Telo

me

re le

ng

th

90-100 70-80

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

Blasco Lansdorp

Blackburn

Harley

Blasco

TRF analysis

Dot blot

Scalability

Flow cytometry

qPCR

Southern Blot

STELA

qFISH

Multiple

sample

processing

Single

sample

processing

Average telomere length Percentage of short telomeres

HT qFISH (blood)

Telomapping (tissue)

2009 Nobel Prize Winner for the Discovery of

Telomerase – Dr. Carol Greider

Fluorescence In Situ Hybridization (FISH) for

Measuring Telomeres

• Q-FISH or quantitative fluorescence in situ hybridization

FISH provides a sensitive method for specific telomere length detection

short telomeres long telomeres

DAPI signal in blue (chromosomes)

CY3 signal in yellow (telomeres)

HT Q-FISH Telomere Length Measurements

Canela & Vera et al., PNAS, 2007

0 5 10 15 200

20

40

60

mean telomere length (kb)

freq

uen

cy

Mean TL

% Short telomeres

1) Blood sample 2) HT Q-FISH 3) Confocal microscopy

4) Capture of individual

telomere signals 5) Data processing 6) Data analysis

About one teaspoon

The Percent of Short Telomeres, Not Average

Telomere Length Determines the Onset of Disease

0 10 20 30 40 50 60 700

5

10

15

20

Me

an

te

lom

ere

le

ng

th (

Kb

)

Age (years)

0 10 20 30 40 50 60 700

10

20

30

40

50

60

% s

ho

rt t

elo

me

res

(<

3kb

)

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

Luminal epithelial cells

Stromal fibroblast cells

Telomere FISH

Basal

cells

Alan Meeker

Prostatic

Intraepithelial

Neoplasia (PIN)

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

It’s 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 diseases—including 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

aging and the telomere connection-dr.jerry shay-a4m-orlando-april 2013

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