Dra. María Blasco - El origen de la enfermedad

Aging & the origin
of disease
Telomeres & Telomerase Group
Spanish National Cancer Research Center (CNIO)
Madrid, Spain

AAggiinngg iiss tthhee hhiigghheesstt rriisskk ffaaccttoorr ffoorr mmoosstt ddiisseeaasseess ((iinncclluuddiinngg ccaanncceerr))
HHeeaalltthh ssppaann
4400--5500 yyeeaarrss

……aanndd ccaarrddiioovvaassccuullaarr ddiisseeaasseess ((iiee..,, hheeaarrtt iinnffaarrcctt))
Health
span
Health
span
4400--5500 y yeeaarrss

““Healthy aaggiinngg”” aass oonnee ooff tthhee GGrraanndd SSoocciieettaall CChhaalllleennggeess ooff EEUU
2009
65 years-> 17% population (7 M)
(25% >80 years)
2050 (projection)
65 years-> 30 % population (13 M)
(30% >80 years: 4 M)
FG CSIC Spain (2010)

Understanding tthhee mmoolleeccuullaarr bbaassiiss ooff aaggiinngg ttoo uunnddeerrssttaanndd ddiisseeaassee
genetic &
environmental
biomarkers
for prevention
& prognosis
Infancy/youth/adulthood aging
cancer
cardiovascular
diabetes
renal disease
immunosenescence
neurodegeneration
etc…
cause
consequences
therapeutic
interventions
aging
cancer
cardiovascular
diabetes
renal disease
immunosenescence
neurodegeneration
etc…
Infancy/youth/adulthood

AA cchhaannggee iinn ppaarraaddiiggmmaa
uunnttili l nnooww ffuuttuurree
Partridge

TThhee hhaallllmmaarrkkss ooff aaggiinngg
Lopez-Otín, Blasco, Partridge, Serrano & Kremer, Cell, 2013

TTeelloommeerreess aarree lloosstt eevveerryyttiimmee tthhaatt aa cceellll ddiivviiddeess……
telomere telomere
Chromosome
(parent cell)
“the end-replication problem”
DNA is lost from the ends
Chromosome
(daughter cell #1)
Chromosome
(daughter cell #2)

An eemmbbrryyoonnaarryy ((pplluurriippootteennccyy)) ggeennee kknnoowwnn aass tteelloommeerraassee iiss aabbllee ttoo
eelloonnggaattee tteelloommeerreess aatt tthhee bbllaassttooccyysstt ssttaaggee aanndd rreesseett tteelloommeerree lleennggtthh..
TTeelloommeerraassee aallssoo eelloonnggaatteess tteelloommeerreess iinn iinndduucceedd pplluurriippootteenntt sstteemm ((iiPPSS))
cceellllss
TThhiiss ggeennee,, tteelloommeerraassee,, iiss ssiilleenncceedd aafftteerr bbiirrtthh …… hhoowweevveerr ……
CCaanncceerr cceellllss mmaannaaggee ttoo rreeaaccttiivvaattee tteelloommeerraassee,, tthhuuss eessccaappiinngg tthhee mmoorrttaall
ffaattee ooff aadduulltt cceellllss aanndd bbeeccoommiinngg iimmmmoorrttaall
genes telomeres
genes telomeres
TTAGGG
TTAGGG
AATCCC AATCCC
TTAGGG TTAGGG
AATCCC AATCCC
TTAGGG TTAGGG
AATCCC AATCCC
cell death
cell senescence
aging
TTAGGG AATCCC AATCCC
cancer
TTAGGG TTAGGG
AATCCC AATCCC
TTAGGG TTAGGG
AATCCC AATCCC
TTAGGG TTAGGG
AATCCC AATCCC
TTAGGG TTAGGG
AATCCC AATCCC
TTAGGG TTAGGG
AATCCC AATCCC

SShhoorrtt tteelloommeerreess ccaauussee aaggiinngg iinn mmiiccee
Telomerase-deficient mice (Terc-/-)
Stem cell dysfunction
Decreased regenerative capacity
Premature aging
Less cancer in late generations
Blasco et al., Science (1995)
Blasco, Lee, et al., Cell (1997)
Lee, Blasco, et al., Nature (1998)
González-Suarez et al., Nat Genet (2000)
Flores et al., Science (2005)

TTeelloommeerraassee aass aann aannttii--ccaanncceerr ttaarrggeett ((IImmeetteellssttaatt))

Telomere length as a biomarker with
prognostic value?
Lower percentiles of telomere length=
higher risk of diseases (cardiovascular,
neurodegenerative, death by infections)
cancer
(> 95% of cancers
activate telomerase)
high
Telomere length
TTeelloommeerreess,, tteelloommeerraassee aanndd aaggiinngg
Tissues and organs (including stem cells)
Mutations in telomerase & health
telomere proteins
disease
age
Human pathologies due to telomere defects
(“TELOMERE SYNDROMES”)
dysqueratosis congenita (DKC1, Terc, Tin2)
aplastic anemia (Terc, Tert, Tin2)
idiophatic pulmonary fibrosis (Terc, Tert)
GI tract diseases (Terc,Tert)
loss of the regenerative capacity of the skin
lungs, bone marrow, intestine…

TTeelloommeerreess ppoowweerrffuullllyy qquuaannttiiffyy lliiffee´ss iinnssuullttss
Blackburn Blackburn & & E Eppeel,l ,N Naatuturere, ,2 2001122
# Life habits influence telomere length : smoking, exercise, obesity, nutrition
# Perceived stress as adults shortens telomeres (Epel et al., PNAS, 2004)
# Stress as children and stress suffered by the mother during pregnancy shortens
telomeres (Shalev et al, Mol Psychiatry, 2012; Entringer et al., PNAS, 2011)
# Personality disorders shorten telomeres(Elvsashagen et al., J. Affect. Disord., 2011)

WWee ddeevveellooppppeedd aa hhiigghhtthhrroouugghhppuutt mmeetthhoodd ttoo qquuaannttiiffyy sshhoorrtt tteelloommeerreess
1) Obtaining blood sample (5 ml) 2) HT Q-FISH 3) Confocal microscopy
6) Data analysis 5) Data processing 4) Capture of individual telomeres
Canela et al., PNAS, 2007
0 5 10 15 20
60
40
20
0
mean telomere length (kb)
frequency
Mean TL
% Short telomeres

Current collaborations to aasssseessss tthhee pprrooggnnoossttiicc vvaalluuee ooff tteelloommeerree lleennggtthh
Infarct, CVD
(CNIC, Madrid &
University Hospital,
Salamanca)
Fertility
(IVI, Madrid)
Bipolar & personality
dissorders
(University Hospital, Oslo)
Hepatic transplatation
tolerance
(Hospital Clínic, Barcelona)
AIDS
(Hospital Clínic,
Barcelona)
Toxicity of
breast cancer treatments
(CNIO, Madrid)
Risk of breast/ovarian
cancer (BRCA1/2 carriers)
(Familiar Cancer Consultancy
CNIO, Madrid)

Telomere lleennggtthh iinn ppeerrssoonnaalliizzeedd aanndd pprreevveennttiivvee mmeeddiicciinnee
INHERITED
Genetic tests: genes as risk factors for disease
but do not reflect on the environment or life habits
Telomere length: indication of the degree of
cellular aging (reflect environment, life habits)
INHERITED ENVIRONMENT
MORE POWERFUL PREDICTIONS
OF RISKS AND TIME OF ONSET
DEVELOPMENT OF
NEW TREATMENTS TO
PREVENT DISEASE
DEVELOPMENT OF
NEW TREATMENTS TO
PREVENT DISEASE
# aging is the highest risk factor for all diseases
# telomere length integrates both inheritance & life habits & environmental factors

Short telomeres as preditors ooff oonnsseett ooff hheerreeddiittaarryy bbrreeaasstt && oovvaarriiaann ccaanncceerr
BRCA1/BRCA2 carriers
breast/ovarian cancer
Martínez-Delgado et al., PLoS Genetics, 2011
Martínez-Delgado et al., J. Medical Genetics, 2012
Telomere length
Collaboration with Javier Benítez,
Human Cancer Genetics Group, CNIO
Age
healthy

How to rejuvenate
telomeres to prevent
disease?

telo Does telommeerraassee aaccttiivvaattiioonn ddeellaayy pphhyyssiioollooggiiccaall aaggiinngg??
TERT transgenic mice (K5-TERT)
Wild-type “Triple”
Less aging
Only minor increase in survival owing to slightly more cancer
González-Suarez et al., EMBO J. (2001)
TERT transgenic mice + increased tumor supressor genes
(Sp53+Sp16+Sp19ARF): “triple” mice
Less cancer
Less aging: better glucose tolerance, neuromuscular coordination,
bone density, skin fitness…
Longer telomeres & less DNA damage with aging
40% increase in lifespan
Tomás-Loba et al., Cell (2008)

IInnccrreeaasseedd ““hheeaalltthh ssppaann”” && lloonnggeevviittyy iinn TTRRIIPPLLEE mmiiccee
Overall survival Cancer-free survival
100
80
60
40
20
0
Sp53 (n=25)
Triple (p=0.003; n=6)
0 60 80 100 120 140 160 180
survival (%)
age (weeks)
+50%
Control (n=25)
100
80
60
40
20
0
Sp53 (n=49)
Triple (p<0.001; n=12)
0 60 80 100 120 140 160 180
survival (%)
age (weeks)
+40%
Control (n=49)
60
40
20
0
p=0.003
n=25
n=6
60
40
20
0
p<0.001
n=49
n=12
Control Triple
Survival at 3 years (%)
Control Triple
Survival at 3 years (%)

IImmpprroovveedd hheeaalltthh llaattee iinn lliiffee iinn TTRRIIPPLLEE mmiiccee
Improved neuromuscular fitness
young (5-20 weeks) old (116-160 weeks)
125
100
75
50
25
0
Success rate (%)
p=0.02
n=11
n=9
wildtype
p=1
n=8 n=10
TRIPLE

WWhhaatt iiff wwee wwoouulldd ddoouubbllee hheeaalltthh ssppaann iinn hhuummaannss??
Heath-span
4400 y yeeaarsrs
Average life span (85 years)
Maximum life span (120 years)
survival
health diseases
age
Heath-span
Average life span (115 years)
Maximum life span (120 years ?)
survival
age
intervention
diseases
health
8800 y yeeaarsrs

A gene therapy
of aging
(adeno associated viruses, AAV)

TThheerraappeeuuttiicc ssttrraatteeggyy ffoorr tteelloommeerraassee aaccttiivvaattiioonn
Bernardes de Jesus et al, EMBO Mol Med (2012)
Aging is produced, at least in part, due to telomerase
deficiency in adult life.
We have used gene therapy to deliver TERT late in life (1 & 2
year old mice): gene therapy of aging.
We have used state-of-the art gene therapy vectors (AAV),
currently used in clinical trials owing to their safety: non-integrative,
non immunogenic.
More than 700 individuals have been already treated with
these vectors for different genetic diseases in clinical trials.

CClliinniiccaall ttrriiaallss wwiitthh aaddeennoo aassssoocciiaatteedd vviirruusseess ((AAAAVV))
Mingozzi and High, Nature Reviews Genetics, 2011

A TERT based gene therapy of aging: ssiinnggllee ttrreeaattmmeenntt wwiitthh AAAAVV99--TTEERRTT
Lung
p= 0,03
n=3
AAV9-
mTERT
n=3
AAV9-
eGFP
p= 0,003
n=3
AAV9-
mTERT
n=3
AAV9-
eGFP
~ 1 yr ~2 yr
Fold changes in telomerase activity
AAV9-mTERT #1 AAV9-mTERT #2 AAV9-mTERT #3 AAV9-eGFP #1 AAV9-eGFP #2
Protein (mg) 1 3 5 5 1 3 5 5 1 3 5 5 1 3 5 5 1 3 5 5
Rnase - + - - - + - - - + - - - + - - - + - - - +
Bernardes et al. , EMBO Molecular Medicine, 2012
Lung (2-year old group)
Hela
IC
3 3
3 months PI

1 & 2 year-old mice
better glucose tolerance
better skin fitness
less cognitive decline
less osteoporosis
delayed cancer
Improved neuromuscular
coordination
longer telomeres
lower DNA damage

Single treatment with AAAAVV99--mmTTEERRTT iimmpprroovveess nneeuurroommuussccuullaarr ccoooorrddiinnaattiioonn
p= 0,4 p= 0,01
100 n= 18
80
60
40
20
0
n= 6
n= 8
Tightrope success (%)
n= 11
~1 yr
(6 months PI)
~1 yr
(11 months PI)
AAV9-
mTERT
AAV9-
eGFP
AAV9-
mTERT
AAV9-
eGFP

AA ssiinnggllee AAAAVV99--mmTTEERRTT ttrreeaattmmeenntt llaattee eexxtteennddss mmoouussee lliiffeessppaann
~1 yr old group
13% median lifespan
20% maximum lifespan
V.I. – Viral injection
AAV9-eGFP n=12
AAV9-mTERT n=21
Control n=33
eGFP vs mTERT
p= 0,02 (Log Rank test)
Control vs mTERT
Weeks
Percent Survival
V. I.
24%
13%
24% median lifespan
13% maximum lifespan
Weeks
Percent Survival
V. I.
~2 yrs old group
AAV9-eGFP n=14
13%
20%
eGFP vs mTERT
Control vs mTERT
AAV9-mTERT n=23
Control n=25

SSiinnggllee ttrreeaattmmeenntt wwiitthh AAAAVV99--mmTTEERRTT ddooeess nnoott iinnccrreeaassee ccaanncceerr iinncciiddeennccee
……..iinnddeeeedd,, ccaanncceerr aappppeeaarrss llaatteerr iinn TTEERRTT--ttrreeaatteedd mmiiccee
100
80
Histiocytic sarcoma
Adenoma
Lymphoma
Adenocarcinoma
(%)
affected 60
40
Mice 21 20
0
p= 0,87
n= 26 n= 40
3311
77
22
3344
1144
99
AAV9-eGFP AAV9-mTERT

trea A single treattmmeenntt wwiitthh AAAAVV99--TTEERRTT iinnccrreeaasseess ““hheeaalltthh--ssppaann”” aanndd lloonnggeevviittyy
Commentary in:
Boccardi & Herbig, EMBO Molecular Medicine, 2012

Telomere shortening is one of tthhee ccaauusseess ooff pphhyyssiioollooggiiccaall aaggiinngg && ddiisseeaassee
Infancy/youth/adulthood aging
cancer
cardiovascular
diabetes
renal disease
immunosenescence
neurodegeneration
etc…
Telomere
shortening
consequences
TERT
activation
aging
cancer
cardiovascular
diabetes
renal disease
immunosenescence
neurodegeneration
etc…
Infancy/youth/adulthood

WWee aarree tteessttiinngg TTEERRTT aaccttiivvaattiioonn iinn……
Telomere syndromes
(ie., aplastic anemia)
Age-associated diseases:
cardiovascular,
neurodegenerative…

Longitud telomérica
“Las edades del hombre”
Hans Baldung (aka “Grier”)
Museo del Prado
“Saber” para prevenir
& diagnóstico precoz
TERT

The Telomeres & Telomerase GGrroouupp,, CCNNIIOO,, MMaaddrriidd
Christian Bär Rosa Serrano
Fotografía Amparo Garrido

Funding of Telomeres & TTeelloommeerraassee GGrroouupp aatt CCNNIIOO

The Telomeres & Telomerase GGrroouupp,, CCNNIIOO,, MMaaddrriidd
Christian Bär Aksinya Derevyanko Juanma Povedano Mercedes Gallardo
Bruno Bernardes
Agueda Tejera Maria Garcia Martina Stagno Miguel Foronda Paula Martinez
Ralph Schneider Gina de Bonis Nani Marion
Isabel L. Silanes
Fabian Beier
Elsa Vera
Iole Ferrara
Alicia Lemus
Nora Soberon
Ben Kumfmüller
Elisa Varela
Rosa Serrano
Ianire Garrobo

Effects ooff bbrreeaasstt ccaanncceerr ttrreeaattmmeenntt oonn tteelloommeerree lleennggtthh
AC+T₁: Doxorrubicine/ cyclophosphamide + Paclitaxel
T+AC₂: Paclitaxel+ Doxorrubicine/ cyclophosphamide
T+FEC90₄: Paclitaxel+ (5-Fluoracile/Epirrubicine/ cyclophosphamide)

TTeelloommeerree lleennggtthh hheetteerrooggeenneeiittyy iinn hhuummaann ccaanncceerrss
basal cell carcinoma
long telomeres
short telomeres
Single cell quantitative telomere FISH
Schneider et al., unpublished
(“telomapping”)
Flores et al., G&D, 2008

SShheelltteerriinn:: tthhee pprrootteeiinn ccoommpplleexx tthhaatt pprrootteeccttss cchhrroommoossoommee eennddss
TTAGGG TTAGGG
AATCCC AATCCC
TTAGGG TTAGGG
AATCCC AATCCC
genes telomeres
genes telomeres
TTAGGG
TTAGGG
AATCCC AATCCC
TRF1
TRF2
cell death
cell senescence
aging
Pot1
TPP1
“shelterin”
Pot1
Tin2 TPP1
Rap1
TPP1
Martínez & Blasco, Nature Reviews Cancer, 2011
anti-cancer
targets?

Validation of TRF1 as
an anti-cancer target

TTRRFF11 aass aann aannttii--ccaanncceerr ttaarrggeett
TRF1 is essential for viability of dividing cells
Martínez et al., Genes & Dev., 2009; Beier et al., Blood, 2012; Schneider et al., Nat Com, 2013
TRF1 deletion rapidly uncaps telomeres
Martínez et al., Genes & Dev., 2009
TRF1 deletion induces a persistent DDR
Martínez et al., Genes & Dev., 2009
TRF1 is high in stem cells & required for stemness
Schneider et al., Nature Communications, 2013
We have an eGFP-TRF1 reporter mouse (screens)
Schneider et al., Nat Communications, 2013
Mitotic catastrophe
Chromosome end-to-end fusions
Persistent DDR
Loss of stemness
“green” telomeres

TTeelloommeerree uunnccaappppiinngg aass aann aannttii--ccaanncceerr tthheerraappyy
A mouse model for K-ras induced lung carcinogenesis
TRF1 lox Excised TRF1 (TRF1 Δ)
K-ras LSLG12V
Cre
excision
Activated oncogenic K-ras (K-ras G12V ) + b Gal staining
In vivo tumor follow-up
0 9 12 14 16 18 20 22 24
García-Beccaria et al., submitted
Adeno-Cre
Intratracheal
Inoculation 1st CT 2nd CT 3rd CT 4th CT 5th CT
PET-CT
FDG Sacrifice
Weeks after
inoculation
6th CT
Guerra et al., Cancer Cell (2003)
lung cancer

TRF1 ddeelleettiioonn hhaammppeerrss KK--RRaass iinndduucceedd lluunngg ccaarrcciinnooggeenneessiiss
CT-SCAN
p53+/+
p53-/-

Less and ssmmaalllleerr ccaarrcciinnoommaass iinn TTRRFF11 ddeelleetteedd lluunnggss

tu Less malignant tummoorrss && iinnccrreeaasseedd ssuurrvviivvaall iinn tthhee aabbsseennccee ooff TTRRFF11
PET

TRF1 deletion leads to GG22 aarrrreesstt && eennddoorreedduupplliiccaattiioonn iinn ccaarrcciinnoommaass
Normal
length
telomere
Normal
length
telomere
s
n=22
lesions
8 mice
n=11
lesions
9 mice
**
120
100
80
60
40
20
0
Telomere fluorescence (a.u)
s
Trf1+/+
K-ras+/G12V
p53-/-
Trf1Δ/Δ
K-ras+/G12V
p53-/-

TRF1 deletion leads to telomere damage aanndd aappooppttoossiiss iinn ccaarrcciinnoommaass

TRF1 downregulation iinn cceellllss ddeerriivveedd ffrroomm mmoouussee KK--rraassΔΔ//GG1122VV pp5533--//-- ccaarrcciinnoommaass
Nude mice
stable K-rasΔ/G12V p53-/-
mouse lung tumor-derived cell line
(M.Barbacid’s lab)
shTRF1 Subcutaneous injection/
tail vein injection
(lung metastasis)

TTRRFF11 ddoowwnnrreegguullaattiioonn iimmaappiirrss tthhee ggrroowwtthh ooff ttuummoorrss bbyy cceellllss ddeerriivveedd ffrroomm
KK--rraassΔΔ//GG1122VV pp5533--//-- ccaarrcciinnoommaass
10
8
6
4
2
shTRF1
scrambled
400
300
200
100
*
shTRF1
scrambled
140
120
100
80
60
40
20
0
TRF1 expression
* **
n=3 n=3 n=10 n=6
Control
shTRF1
Control
shTRF1
Injected cells Tumors
0
Control
tumors
shTRF1
tumors
Tumor volume (mm3)
n=10 n=6
0.60
0.40
0.20
0
Tumor weight (g)
p=0.086
n=10 n=6
Control
tumors
shTRF1
tumors
*
Control
tumors
shTRF1
tumors
0
Tumor latency (days)
n=10 n=6

TTRRFF11 ddoowwnnrreegguullaattiioonn iimmaappiirrss tthhee ggrroowwtthh ooff ttuummoorrss bbyy cceellllss ddeerriivveedd ffrroomm
KK--rraassΔΔ//GG1122VV pp5533--//-- ccaarrcciinnoommaass bbyy iinndduucciinngg DDNNAA ddaammaaggee,, aappooppttoossiiss……
Ki67 γH2AX CA3
800
600
400
200
0
n=10 n=6
Control
tumors
Ki67- positive cells/ field
shTRF1
tumors
***
Control
tumors
shTRF1
tumors
10
8
6
4
2
0
AC3- positive cells/ field
**
n=10 n=6
Control
shTRF1
ɣ-H2AX
Giant nucleus Anaphase bridge
160
n=10 n=6
H2AX- positive cells/ field Control
120
80
40
0
tumors
shTRF1
tumors
*

TRF1 downregulation iimmppaaiirrss tthhee ffoorrmmaattiioonn ooff lluunngg mmeettaassttaassiiss bbyy cceellllss ddeerriivveedd
1.6
1.2
0.8
0.4
0
***
Control
metastasis
shTRF1
metastasis
Metastasis area (mm2)
n=447 n=413
ffrroomm KK--rraassΔΔ//GG1122VV pp5533--//-- ccaarrcciinnoommaass
Control 5000 μm shTRF1
Control
DAPI Trf1
Tumor
Tumor Bronchius
Bronchius
shTRF1

TTRRFF11 ddoowwnnrreegguullaattiioonn iimmaappiirrss tthhee ffoorrmmaattiioonn ooff mmeettaassttaassiiss bbyy cceellllss ddeerriivveedd ffrroomm
KK--rraassΔΔ//GG1122VV pp5533--//-- ccaarrcciinnoommaass bbyy iinndduucciinngg DDNNAA ddaammaaggee,, aappooppttoossiiss……
80
60
40
20
0
H2AX- positive cells/ field
n=7 n=6
Control met shTRF1
metastasis
***
***
n=7 n=6
Control met shTRF1
metastasis
800
600
400
200
0
Ki67- positive cells/ field
Ki67 γH2AX CA3
*
8
6
4
2
0
Control met shTRF1
metastasis
AC3- positive cells/ field
n=7 n=6

TTRRFF11 ddoowwnnrreegguullaattiioonn iinn cceellllss ddeerriivveedd ffrroomm hhuummaann KK--rraass mmuuttaatteedd lluunngg ccaanncceerr
Nude mice
A549 stable human lung tumor-derived
cell line
shTRF1
Subcutaneous injection
7 11 13 15 17 19 21 24 26
4
3
2
1
0
A549 shScrambled (n=8)
A549 shTRF1 (n=8)
Tumor diameter (mm)
*

Transient systemic TTRRFF11 ddeelleettiioonn iinn aadduulltt mmoouussee ttiissssuueess:: ddeelleetteerreeoouuss eeffffeeccttss??
Trf1+/+ (n=4)
Trf1Δ/Δ (n=4)
* ** ** * * *** ** * * ***
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
HEART
INTESTINE
LUNG
LIVER
KIDNEY
BONE MARROW
SKIN
BLOOD
BRAIN
STOMACH
Trf1 levels
Trf1lox/lox
Ubiquitin-CreERT2+/T
Tamoxifen diet
7 weeks
Trf1 is sucessfully
downregulated in all
the tissues studied

Transient TRF1 deletion iinn aadduulltt mmoouussee ttiissssuueess ddooeess nnoott iimmppaaiirr mmoouussee vviiaabbiilliittyy
n.s
Tamoxifen diet start
point
Mice with the
phenotype
Trf1+/+ Trf1Δ/Δ
SKIN -Low cellularity of basal
layer
-Hair follicle cysts
0/4
0/4
3/4
3/4
INTESTINE -Atrophy of microvilli
-Increased mitotic
figures
0/4
0/4
0/4
4/4
BONE
MARROW
-Moderate aplasia
-Megacaryocytes with
reduced cytoplasm
0/4
0/4
1/4
4/4

TTrraannssiieenntt TTRRFF11 ddeelleettiioonn iinn aadduulltt mmoouussee ttiissssuueess ddooeess nnoott iimmppaaiirr ttiissssuuee ffuunnccttiioonn
Transient Trf1 systemic abrogation generates minor histological alterations in
Trf1+/+ Trf1Δ/Δ
200 μm 200 μm
SKIN
Normal epithelium and
hair follicle
Hair follicle cyst and
anisocaryosis
100 μm 100 μm
Low cellularity in basal skin
and normal hair follicle
100 μm
the skin (ie., low cellularity)

Trf1+/+ Trf1Δ/Δ
200 μm 200 μm
INTESTINE
Increased mitotic
figures. normal nuclei
Giant nuclei
50 μm 50 μm
n.s
Trf1+/+ Trf1Δ/Δ
450
350
250
150
50
microvilli length (pixels)
the gut (ie. normal microvilli, aberrant nuclei, increased mitosis)

BM, that do not compromise viability nor tissue function
Trf1+/+ Trf1Δ/Δ
400 μm 400 μm
*
50 μm 50 μm
BONE MARROW
50 μm
BONE MARROW
Moderate aplasia and
platelets with small cytoplasm
Normal cellularity and platelets Normal cellularity and platelets
* n.s n.s

The eeffffeeccttss ooff TTRRFF11 aabbrrooggaattiioonn iinn aadduulltt ttiissssuueess aarree ttrraannssiieenntt
Trf1 expression and blood cell populations recover after tamoxifen removal
10000
5000
1200
800
10
5
n.s
Lymphocytes
Granulocytes
n.s
Erythrocytes
Platelets
0
+TMX Trf1+/+ (n=13)
+TMX Trf1lox/lox (n=13)
-TMX Trf1lox/lox (n=3)
Cells (109/L)
n.s
n.s
n.s
*
* *
BLOOD INTESTINE SKIN B.MARROW
300
200
40
30
20
10
0
+TMX Trf1lox/lox (n=4)
-TMX Trf1lox/lox (n=3)
Trf1 expression
*
**
*
*

screening for drugs
that disrupt TRF1
binding to telomeres

TTRRFF11 rreeppoorrtteerr mmoouussee:: TTRRFF11--eeGGFFPP ““kknnoocckk--iinn”” mmiiccee
TRF1 endogenous locus
ATG
EGFP
ATG
Exon1
Exon2
Exon1
Exon2
neo
eGFP-TRF1 KI
Neo T
Inframe linking sequence loxP loxP
EGFP
eGFP-TRF1 KI
Neo -
Inframe linking sequence loxP
Recombinational
knock-in
+CRE
F R
eGFP-TRF1
+/+ +/KI KI/KI
eGFP-TRF1
endogenous TRF1
live visualization of telomere foci (eGFP) in a single cell nucleus

HT screening to identify compounds tthhaatt ddiissrruupptt TTRRFF11 tteelloommeerree bbiinnddiinngg
IN COLLABORATION WITH EXPERIMENTAL THERAPEUTICS PROGRAM CNIO
Screening using eGFP-Trf1ki/ki Induced Pluripotent Stem Cells (iPS)
(very high eGFP-TRF1 levels)
eGFP-Trf1 DAPI
eGFP-Trf1ki/ki
Sh-scramble ShTrf1
Positive
Control
Inhibition
Control
Treatment with
chemical library
(640
compounds)
Opera High Content
Screening system
Quantification by
High-speed
image
Analysis
Méndez et al., unpublished

HT screening to identify ccoommppoouunnddss tthhaatt ddiissrruupptt TTRRFF11 tteelloommeerree bbiinnddiinngg
IN COLLABORATION WITH EXPERIMENTAL THERAPEUTICS PROGRAM CNIO
Quantification setups
eGFP-Trf1ki/ki
LI HI LI: Low Intensity
Cut-off
Intensity (a.u.f.)
Q1 Q2 Q3
Foci with intensity ≤ cut-off
HI: High Intensity
Foci with intensity ≥ cut-off
Quartile (Q) distribution
Proof of concept
Z factor (0.5-1): high reproducibility
eGFP-Trf1ki/ki vs eGFP-Trf1ki/ki
Z**=0.75
eGFP-Trf1ki/ki vs shTrf1
Z**=0.86
Identification of hit compounds inhibiting Trf1 binding to telomeres
Control ETP-
47228
ETP-
47037
1.2
1
0.8
0.6
0.4
0.2
0
DMSO sh-TRF1 ETP-
47228
ETP-
47037
Mean e-GFP-Trf1 foci
intensity (a.u.f)
***
***
**

HT screening to identify compounds tthhaatt ddiissrruupptt TTRRFF11 tteelloommeerree bbiinnddiinngg
Mechanism of action: do compounds affect TRF1 binding to the
FRAP: fluorescence recovery after photobleaching of eGFP-TRF1 in
living cells
E. Varela
% and time of recovery
telomere in vivo?

E. Varela
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
FRAP oonn iinnddiivviidduuaall tteelloommeerreess ooff eeGGFFPP--TTRRFF11 iiPPSS cceellllss
0 50 100 150 200 250 300 350 400
DMSO, 11 movies
47228, 11 movies
50946, 14 movies
47361, 12 movies
Time (s) Relative fluorescence intensity

Cellular eeffffeeccttss ooff TTRRFF11 iinnhhiibbiittoorrss oonn iiPPSS cceellllss ((EETTPP--4477222288))

Cellular effects of TRF1 inhibitors oonn lluunngg ccaanncceerr cceellllss ((EETTPP--4477222288))
DMSO ETP-47228 ETP-47037
1.2
1
0.8
0.6
0.4
0.2
0
DMSO ETP-
47228
ETP-
47037
Mean Trf1 foci intensity
(a.u.f)
*
*
TRF1
4
3
2
1
0
DMSO ETP-
47228
ETP-
47037
Mean gH2AX nuclear
intensity (a.u.f)
**
***
25
20
15
10
5
0
DMSO ETP-
47228
ETP-
47037
Cells with ≥ 3 TIFs (%)
**
***
gH2AX
RAP1 + gH2AX

Cellular effects of TRF1 inhibitors oonn lluunngg ccaanncceerr cceellllss ((EETTPP--4477222288))
1.2
1
0.8
0.6
0.4
0.2
0
ETP-47228
ETP-47037
0.0
0.01
0.02
0.07
0.21
0.62
1.85
5.56
16.67
50.01
Relative growth
normalized to DMSO
Concentration (mM)
Tumor diameter change
(x-fold)
*
DMSO ETP-47228

EETTPP--4477003377 ttrreeaattmmeenntt ssttooppss tthhee ggrroowwtthh ooff ssttaabblliisshheedd lluunngg ccaarrcciinnoommaass
p=0.058
Vehicle
(10 days)
ETP-47037
(10 days)
Tumor area change
post-treatment (x-fold)
CT CT
Sacrifice
Histological & Molecular
analysis
0 1 2 3 4 5 6 7 8 9 10
Days
ETP
47037
ETP-47037 vehicle
Day 0 Day 10

12
0
10
0
40
60
80
20
0
Mean foci intensity (a.u.f) (%)
EETTPP--4477003377 ttaarrggeettss TTRRFF11 iinn vviivvoo
Untreated
ETP-470037
*** ***
Intestine Lung tumors
ETP-470037 Untreated
Untreated ETP-470037
Lung tumors Intestine
Normal mitosis
Giant multinucleated
Micronuclei

In conclusion: it is possible to ttaarrggeett sshheelltteerriinn pprrootteeiinnss
We targeted telomere “capping” by inhibition of TRF1
Deletion of TRF1 in lung cancer mouse models impairs lung
cancer and metastasis, independently of telomere length
Transient systemic deletion of TRF1 in adult mice does not
affect organismal viability and only has minor defects in
tissues, which are corrected upon release fromTRF1 inhibition
We found small molecules that dysrupt TRF1 binding to
telomeres in vivo
TRF1 inhibitors induce DNA damage at telomeres and impair
proliferation of iPS cells and cancer cell lines

Telomere length &
species longevity?

Telomere length aatt ddaayy 2255 ooff aaggee pprreeddiiccttss lliiffeessppaann iinn bbiirrddss

FFiirrsstt lloonnggiittuuddiinnaall tteelloommeerree lleennggtthh aannaallyyssiiss tthhrroouugghhoouutt tthhee lliiffeessppaann ooff mmiiccee
1) Obtaining blood sample (300μl) 2) HT Q-FISH 3) Confocal microscopy
4) 6) Data analysis 5) Data processing Capture of individual telomeres
Vera et al., Cell Reports,
2012
Facial vein bleeding
0 5 10 15 20
60
40
20
0
frequency
Mean TL
% Short telomeres

Mouse tteelloommeerreess sshhoorrtteenn 110000--ttiimmeess ffaasstteerr tthhaann hhuummaann tteelloommeerreess
Humans: 70 bps/year
Mice: 7 Kb/year
Linear regression:
WT: Slope=-7.03 ± 1.24 kb/year
Linear regression:
WT: Slope=-7.03 ± 1.24 kb/year
R2=0.31 (Quadratic: R2=0.34)
p<0.0001
R2=0.31 (Quadratic: R2=0.34)
p<0.0001
TgTERT: Slope=-7.05 ± 1.60 kb/year
TgTERT: Slope=-7.05 ± 1.60 kb/year
R2=0.22 (Quadratic: R2=0.30)
p<0.0001
R2=0.22 (Quadratic: R2=0.30)
p<0.0001
80
70
60
50
40
30
20
10
0
0.0 0.5 1.0 1.5 2.0 2.5
Age (years)
Mean telomere length (kb)
TThhee iinnccrreeaassee iinn tthhee %% ooff sshhoorrtt tteelloommeerreess pprreeddiiccttss iinnddiivviidduuaall mmoouussee lloonnggeevviittyy
WT: Slope=-0.005022 ± 0.002550
TgTERT: Slope=-0.01267 ± 0.005604
Vera et al., Cell Reports,
2012
short telomeres (per month)
Increase in the % of
0 50 100 150 200
1.5
1.0
0.5
0.0
-0.5
Lifespan (weeks)
WT: Slope=-0.005022 ± 0.002550
R2=0.2
p=0.06
R2=0.2
p=0.06
TgTERT: Slope=-0.01267 ± 0.005604
R2=0.3
p=0.04
R2=0.3
p=0.04
WT
TgTERT
WT
TgTERT

h Development & validation of a hiigghhtthhrroouugghhppuutt mmeetthhoodd ttoo qquuaannttiiffyy sshhoorrtt tteelloommeerreess
Canela et al., PNAS (2007)
HT-QFISH
Median telomere length (Kb)
≤20 21-30 31-40 41-50 51-60 >60
n= 16 n= 45 n= 78 n= 74 n= 32 n= 35
n= 16 n= 45 n= 78 n= 74 n= 32 n= 35
Age (years)
Q1 Q2 Q3 Q4
15
60
50
10
40
30
5
20
10
0
% short telomeres (<3kb)
≤20 21-30 31-40 41-50 51-60 >60
Age (years)
0
TThhee ppoowweerr ooff sshhoorrtt tteelloommeerreess……
SSnnyyddeerr ccaarrrriieess aa TTEERRTT mmuuttaattiioonn aassssoocciiaatteedd
ttoo aappllaassttiicc aanneemmiiaa
Q5 Q6 Q7 Q8 Chen et al., Cell ( 2012)

AAV9-TERT treated mice show lloonnggeerr tteelloommeerreess iinn ccaarrddiioommyyooccyytteess
Left ventricle infarct remote myocardium (cardiomyocytes) cardiomyocyte TL
200
150
100
50
0
p<0.0001
mean 52.33 a.u.
(1850 nuclei)
mean 64.98 a.u.
(1968 nuclei)
mean spot intensity /
nucleus (a.u.)
AAV9-empty (n=4)
AAV9-Tert (n=4)
DAPI / TEL-CY3 DAPI / TEL-CY3
Q-FISH – qualitative fluorescence in situ hybridisation
Bär/Bernardes, submitted

Tert mRNA expression is also rreepprreesssseedd iinn tthhee hheeaarrtt tthhee ffiirrsstt wweeeekk aafftteerr bbiirrtthh
Tert mRNA expression level
1.5
(relative to day 1)
p=0.034
p=0.016
n=3
n=2
n=3
n=3
1 3 7 10
1.0
0.5
0.0
postnatal day

Within tthhee aadduulltt hheeaarrtt,, AAAAVV99--TTEERRTT ssppeecciiffiiccaallllyy ttrraannssdduucceess ccaarrddiiaacc mmyyooccyytteess
80
60
40
20
0
n=2
n=2
Cardiomyocytes
Cardiac fibroblasts
% eGFP positive cells
Cardiomyocytes (AAV-eGFP 5x1011vg/mouse)
DAPI GFP
MHC Merge

TTeelloommeerraassee aass ttrreeaattmmeenntt ttoo pprreevveenntt hheeaarrtt ffaaiilluurree uuppoonn iinnffaarrcctt
Telomere
shortening
Infancy/youth/adulthood aging Heart infarct
TERT
activation
Increase
survival upon
heart infarct

Treatment of
“telomere syndromes”

SShheelltteerriinn:: tthhee pprrootteeiinn ccoommpplleexx tthhaatt pprrootteeccttss cchhrroommoossoommee eennddss
TTAGGG TTAGGG
AATCCC AATCCC
TTAGGG TTAGGG
AATCCC AATCCC
genes telomeres
genes telomeres
TTAGGG
TTAGGG
AATCCC AATCCC
TRF1
TRF2
cell death
cell senescence
aging
Pot1
TPP1
“shelterin”
Pot1
Tin2 TPP1
Rap1
Epithelial abnormalities are characteristic of
“telomere syndromes”:
Skin hyperpigmentation
Nail dystrophy
Oral leukoplakia
More skin cancer
TPP1 is also necessary for telomerase binding to telomeres
(Tejera, Stagno d´Alcontres et al., Dev. Cell, 2010)
TPP1
Martínez & Blasco, Nature Reviews Cancer, 2011

We generated conditional mmoouussee mmooddeellss ffoorr TTRRFF22,, TTRRFF11,, TTPPPP11,, TTIINN22 aanndd RRAAPP11
hyperpigmentatio
n
Martínez et al., Genes & Dev (2009)
Tejera, Stagno et al., Dev Cell (2010)
Martínez et al., Nature Cell Biology (2010)
Martinez et al., Cell Reports (2013)
Martínez et al., Aging Cell (2014)
TRF1Δ⁄Δ K5-Cre
hyperpigmentation
All dead by 6 days
TRF1
TPP1 Δ⁄Δ K5-cre
All dead by 9 days
Tpp1
Pot1
Rap1D/D K5-Cre
normal survival
obesity & metabolic
syndrome
WT
RAP1
TRF1
TRF2
Tpp1
Pot1
RAP1 Tin2 TRF2
TRF2Δ⁄Δ K5-Cre
All dead by 1 day
Tin2
Tinf2 D/D-K5-Cre
All dead by 1 day

Rap1 bbiinnddss ttoo eexxttrraa--tteelloommeerriicc ssiitteess && rreegguullaatteess ttrraannssccrriippttiioonn
…ooff kkeeyy mmeettaabboolliicc ggeenneess,, aammoonngg ootthheerrss……
Transcriptional regulation
Rap1+/+ Rap1-/-
PPARa
PGC1a
Martínez et al., Nat Cell Biol, 2010
Martínez et al., Nat Rev Cancer, 2011
Martínez et al., Cell Reports, 2013
Commentary by Duong & Sahin, Cell Reports, 2013
ChIP-Seq analysis

Permanent TRF1 deletion in mouse tissues lleeaaddss ttoo lloossss ooff ttiissssuuee
hhoommeeoossttaassiiss && rreeccaappiittuullaatteess tthhee ppaatthhoollooggiieess ooff tteelloommeerree ssyynnddrroommeess
TRF1Δ/Δ
Lack of hair follicles
Lack of sebaceus glands
Hyperpigmentation
Hyperkeratosis
Leukoplakia
Nail distrophy
TRF1flox/flox K5-Cre
skin
TRF1flox/flox Mx1-Cre
Beier et al., Blood (2012)
BM failure
Cellular senescence
BM Short telomeres
TRF1lox/lox; Villin-CreERT2+/T
Schneider et al., Nature Communications (2013)
small
intestine
Colapse of villi/crypts
Apoptosis at SCs
Endoreduplication at SCs
lung
TRF1Δ/Δ KFP+/T SPC-CreERT2 +/T
Pulmonary fibrosis
Povedano et al., unpublished

TRF1 deletion in the BM as aa mmooddeell ffoorr DDKKCC && aappllaassttiicc aanneemmiiaa
……aanndd ffoorr tteessttiinngg tthheerraappeeuuttiicc tteelloommeerraassee aaccttiivvaattiioonn
0 5 10 15
50
40
30
20
10
Telomere length in kb
Time (w)
TRF1flox/flox
Mx1-wt (n=6)
TRF1flox/flox
Mx1-Cre
(n=8)
0 1 2 3
25
20
15
10
5
0
Survival (w) after last measurement
Percentage of telomeres <15 kb
r= -0.80
p=0.02
20
15
10
5
p=0.61
n=8
p=0.04
n=6
n=6
n=5
p=0.37
n=8
n=6
0 1 2 3
30
20
10
0
r=0.90
p=0.002
Survival (w) after last measurement
Telomere length in kb
Telomeres <15 kb
HT-Q-FISH
0
Week 0 Week 4 Week 8
Percentage of telomeres <15 kb
TRF1flox/flox
Mx1-wt
TRF1flox/floxM
x1-Cre
Fabian Beier

AAV9-TERT treatment as a therapeutic ssttrraatteeggyy ffoorr BBMM aappllaassiiaa
Christian Bär
TRF1
deletion

AAV9-TERT ttrreeaattmmeenntt iiss eeffffeeccttiivvee iinn ddeellaayyiinngg ddeeaatthh bbyy aappllaassttiicc aanneemmiiaa
n=4
AAV9-Tert
survival
AAV9-empty n=16
Percent p=0.0006 (Log-rank)
0 20 40 60 80 100
Christian Bär
n=31
p=0.0025 (Log-rank)
87%
55%
n=36
0 20 40 60 80 100
16
AAV9-empty
100
80
60
40
20
0
AAV9-Tert
AA related
other
% AA related death
100
80
60
40
AAV9-Tert
AAV9-empty
Days after AAV treatment
100
80
60
40
20
20
15
10
5
AAV9-Tert
AAV9-empty
0
number of AA deaths
n=4
n=16
0
Days after AAV treatment
n=31 n=36
4
27
20

TTeelloommeerree lleennggtthh aass aa bbiioommaarrkkeerr ooff ttooxxiicciittyy ttoo ttrreeaattmmeennttss
Sporadic breast cancer cases cancer treated (T) with taxanes (n=242)
Ti 6 m Tf 1.5 years post-T
Telomere length

AAV9-empty
AAV9-Tert
Sham (no MI)
500
400
300
200
100
0
MMP9 (ng/ml)
TTEERRTT ttrreeaattmmeenntt rreevveerrttss sseerruumm bbiioommaarrkkeerrss aalltteerreedd uuppoonn MMII
MDC 1860 1600 1340 1250 1030 980 878 1180 4200 3960 3560 3180 1540 1120 1360 852 3650 4200 3590 2430
MCP-5 25 27 28 19 22 16 27 22 26 43 32 42 22 34 7 28 37 24 33 49
MIP-3 beta 6300 4900 5900 7400 6700 4900 5900 5400 7900 6900 7500 6600 7500 4300 4800 4200 7000 12000 8800 7300
IL-18 27000 27000 27000 27000 25000 27000 24000 26000 31000 26000 27000 29000 28000 26000 21000 26000 29000 27000 27000 31000
Ia
HP 159000 159000 158000 156000 156000 153000 162000 158000 150000 158000 156000 157000 148000 166000 156000 160000 155000 153000 135000 154000 Ib
MMP-9 5 196000 199000 157000 187000 187000 182000 174000 235000 458000 229000 223000 177000 249000 182000 189000 270000 235000 273000 368000 II
EGF 29 48 23 41 41 35 35 0 41 54 41 48 41 54 26 73 92 41 29 29
M-CSF-1 8200 9200 9900 9600 10000 8300 8700 8100 11000 11000 8600 11000 8600 11000 9800 10000 8700 9700 10000 12000
LIF 715 715 715 854 646 715 578 578 1210 1140 1170 1070 889 646 715 445 1710 1350 1280 924
MCP-3 177 180 175 211 173 146 165 146 152 187 177 212 156 193 139 149 459 330 284 591
TIMP-1 1500 1700 2400 1600 3300 1700 2100 1400 2600 1500 1500 1900 2200 2400 1700 2100 2800 3500 3500 5700
PAI-1 600 440 540 530 710 310 590 300 880 400 570 420 1400 490 380 380 980 3000 1500 1200
MCP-1 98 88 132 81 110 68 125 79 111 120 100 112 71 105 91 72 130 113 124 251
LNT 681 574 471 380 503 473 507 389 533 550 466 488 385 335 331 268 822 666 545 485
MIP-2 21 47 44 41 29 16 49 24 30 21 22 12 41 12 29 11 32 31 41 35
100
80
60
40
20
0
EGF (pg/ml)
35
30
25
20
15
IL-18 (ng/ml)
3
2
1
0
Fold change mRNA level
ANP
n=5
n=9
0 50 100
IV
V
EMPTY TERT SHAM
III
14600
LTN
Myriad Rodent Multi-Analyte Profile
(array of 60 serum markers)
remodeling cardio protection inflammation fetal program

h Expression of TERT in the adult mouse heeaarrtt ddooeess nnoott aalltteerr hheeaarrtt mmoorrpphhoollooggyy
No virus
AAV9-Tert
TERT
GAPDH
300
250
200
150
0 1 2 3 4 5
Fold change in Tert
mRNA levels
p= 0.003
n=6
n=6
6
4
2
0
TERT fold change
(relative to GAPDH)
p= 0.035
n=2
n=2
no virus
AAV9-empty
AAV9-Tert
SHAM (no MI)
AAV9-Tert AAV9-empty no virus
3
2
1
0
3
2
1
0
Fold change in b-Mhc
mRNA levels
p= 0.6
n=5 n=9
AAV9-empty
AAV9-Tert

Gene expression post MI in TERT ttrreeaatteedd hheeaarrttss rreefflleeccttss lloowweerr iinnffaarrcctt sseevveerriittyy
Cell Cycle
DNA Repair
Inflammation
Transcription
AAV9
empty
AAV9
Tert
Bär/Bernardes, unpublished

Does telomerase re-aaccttiivvaattiioonn aatt tthhee ttiimmee ooff iinnffaarrcctt pprreevveenntt hheeaarrtt ffaaiilluurree??
AAV9-Tert
AAV9-empty
Adult mice
(12 month old)
X
MI
6-weeks survival,
cardiac function
parameters,
heart pathology
X

Telomerase aaccttiivvaattiioonn aatt tthhee ttiimmee ooff iinnffaarrcctt pprreevveennttss hheeaarrtt ffaaiilluurree
25
20
15
10
5
55%
39%
30%
n=33
n=18
n=44
p=0.047
0 10 20 30 40
100
80
60
40
20
empty+no virus
tert
DN-Tert
0
AAV9-empty
AAV9-Tert
Ejection fraction %
p=0.045
n=17
n=17

How to dysrupt
telomere “capping”
to kill cancer cells?

Telomere proteins
as cancer targets?

TRF1 deletion hhaass oonnee ooff tthhee mmoorree sseevveerree ““uunnccaappppiinngg”” pphheennoottyyppeess
Wild type
Knock-out
Trf1 Tpp1 Rap1
5
4
3
0.5
0
End-to-end fusions
(telomere uncapping)
TRF2 Δ/Δ LT-Cre

PPeerrssiisstteenntt DDNNAA ddaammaaggee--ssiiggnnaalliinngg uuppoonn TTRRFF11 ddeelleettiioonn iinn aannyy ttiissssuuee ((SSKKIINN)
wildtype TRF1D/D K5-Cre
epidermi
s
epidermis
dermis
epidermis
dermis
epidermis
dermis
g-H2AX + DAPI
53BP1 + DAPI
g-H2AX + 53BP1 + DAPI
epidermis
dermis
epidermis
dermis
epidermis
dermis
g-H2AX + DAPI
53BP1 + DAPI
g-H2AX + 53BP1 + DAPI
Newborn skin

In conclusion: it is possible to ttaarrggeett sshheelltteerriinn pprrootteeiinnss
We targeted telomere “capping” by inhibition of TRF1
Deletion of TRF1 in lung cancer mouse models impairs lung
cancer and metastasis
Transient systemic deletion of TRF1 in adult mice does not
affect organismal viability and has minor defects in tissues,
which are corrected upon release fromTRF1 inhibition
We found small molecules that dysrupt TRF1 binding to
telomeres in vivo
We are studying these compounds

h Development & validation of a hiigghhtthhrroouugghhppuutt mmeetthhoodd ttoo qquuaannttiiffyy sshhoorrtt tteelloommeerreess
Canela et al., PNAS (2007)
HT-QFISH
Median telomere length (Kb)
≤20 21-30 31-40 41-50 51-60 >60
n= 16 n= 45 n= 78 n= 74 n= 32 n= 35
n= 16 n= 45 n= 78 n= 74 n= 32 n= 35
Age (years)
Q1 Q2 Q3 Q4
15
60
50
10
40
30
5
20
10
0
% short telomeres (<3kb)
≤20 21-30 31-40 41-50 51-60 >60
Age (years)
0
TThhee ppoowweerr ooff sshhoorrtt tteelloommeerreess……
SSnnyyddeerr ccaarrrriieess aa TTEERRTT mmuuttaattiioonn aassssoocciiaatteedd
ttoo aappllaassttiicc aanneemmiiaa
Chen et al., Cell ( 2012) Q5 Q6 Q7 Q8

o Pot1, a component off sshheelltteerriinn,, iiss mmuuttaatteedd
iinn CChhrroonniicc LLyymmpphhooccyyttiicc LLeeuukkeemmiiaa ((CCLLLL))
Carlos López-Otín Elías Campo
Miguel Foronda
Spanish Participation in the International Cancer Genome Consortium

FFiirrsstt ddeessccrriippttiioonn ooff sshheelltteerriinn mmuuttaattiioonnss iinn hhuummaann ccaanncceerr ((CCLLLL))
POT1
DNA binding domain
Q94R
M1L Y36N Y66* K90E Q94R Y223C S250* H266L G272V C591W
OOBB11 OOBB22
K39E I78T R137C G205D G274E L343F P475L
Splicing
N24S
NOTCH1 ~12% of CLL patients
SF3B1 ~10% of CLL patients
POT1 ~9% of CLL IGHV-unmutated patients
NOTCH1 ~12% of CLL patients
SF3B1 ~10% of CLL patients
POT1 ~9% of CLL IGHV-unmutated patients
127 exome seq
214 Sanger seq
12 different mutations (3,5%)
9/12 in the OB folds (ssDNA binding)
Prediction truncated protein
Ramsay*, Quesada*, Foronda* et al., Nature Genetics (2013)
Highlighted in Nature Reviews Cancer, “Gimme Shelter”
(C2h0a1n3g), S. “News & views”, Nature Genetics, (2013)

Mutations in Pot1 iinn CChhrroonniicc LLyymmpphhooccyyttiicc LLeeuukkeemmiiaa ((CCLLLL))
Human
Rat
Mouse
Opossum
Chicken
Frog
Conservation
36
F K P P Y L S K G
F K P P Y L S K G
F K P P Y V S K G
F K P P Y Q S R G
F K P P Y I S K G
F K P P Y R S K G
90 94
K I Q V Y K K E T Q G I T
K I Q V Y K N E L Q G I N
K I Q V Y K N E L Q G I N
K I Q Q Y K N E I Q G V T
K I R E Y N G Q M Q G I T
K I Q K F N D E I Q G I N
223
D I L V Y D N H V
D I L V Y D N H V
D I L V Y D N H V
D I L V Y D N H V
D V L V Y D N H V
D V L V Y D N H V
266 272 591
F H L H G G T S Y G R G I
F H L H G G T C Y G R G I
F H L H G G T C Y G R G I
F H L H G G T C F G R G I
M D M F P P G I
M D M I P P G I
M D M I P P G I
M Y M L P P G K
M N T L P P G R
M D T L P P R K
C
C
C
C
S
C
Y223
H266
K90
Q94
Y36
Affect
conserved
residues
Predicted to
affect
DNA binding

MMuuttaattiioonnss iinn PPoott11 iinn CCLLLL aacctt aass ddoommiinnaanntt nneeggaattiivveess
Normal binding
to telomeres
ChIP
Defective
ssDNA binding Abnormal telomere elongation
Q-FISH

Pot1 mutations ccaauussee tteelloommeerree ffuussiioonnss && ffrraaggiilliittyy iinn cceellllss
Sister chromatid fusions
Telomere fragility (MTS)

PPoott11 mmuuttaattiioonnss ccaauussee tteelloommeerree ffuussiioonnss && ffrraaggiilliittyy iinn CCCCLL ppaattiieennttss
Patient samples
POT1 mut Control
Sister chromatid fusions

PPoott11 mmuuttaattiioonnss && ddiisseeaassee pprrooggrreessssiioonn iinn CCCCLL ppaattiieennttss
Chang, S. “News & views”, Nature Genetics, (2013)

TRF1 deletion in mouse tissues at early developmental stages lleeaaddss ttoo lloossss ooff ttiissssuuee
hhoommeeoossttaassiiss && rreeccaappiittuullaatteess tthhee ppaatthhoollooggiieess ooff tteelloommeerree ssyynnddrroommeess
TRF1Δ/Δ
Lack of hair follicles
Lack of sebaceus glands
Hyperpigmentation
Hyperkeratosis
Leukoplakia
Nail distrophy
TRF1flox/flox K5-Cre
skin
TRF1flox/flox Mx1-Cre
BM failure
Cellular senescence
BM Short telomeres
TRF1lox/lox; Villin-CreERT2+/T
Schneider et al., Nature Communications (2013)
small
intestine
Colapse of villi/crypts
Apoptosis at SCs
Endoreduplication at SCs
lung
TRF1Δ/Δ KFP+/T SPC-CreERT2 +/T
Pulmonary fibrosis
Povedano et al., unpublished

Chromatin
EGFP-TRF1
488nm 100%
output laaser
Bleach point
Fluorescence Recovery
FRAP
Fluorescence Recovery
• Immobile
Fraction
• Mobile
Fraction
binding kinetics of TRF1=(t1/9.4±1.3 s)
70-80% recovery (Plato)
E. Varela
FFlluuoorreesscceennccee rreeccoovveerryy aafftteerr pphhoottoobblleeaacchhiinngg ((FFRRAAPP))

IInntteessttiinnaall cceellllss eexxpprreessssiinngg sstteemm cceellll mmaarrkkeerr LLGGRR5 aarree TTRRFF11hhiigghh
avg. TRF1 fluorescence
per nucleus (a.u.f.)
8
6
4
2
0
p<0.0001
164 cells
n=19
p<0.0001 p<0.0001
196 cells
n=19
139 cells
n=19
712 cells
n=18
370 cells
n=18
360 cells
n=19
All paneth
cells
Lgr5-GFP- Lgr5-GFP+ +4 to+7
Villi
cells
TA
cells
p=0.0360
p=0.6
p<0.0001 p<0.0001
Dapi
TRF1
Lgr5-GFP
Dapi
TRF1
Lgr5-GFP
TRF1
Lgr5-GFP
villi; transient amplifying (TA) cells; +4 to +7 cells;;
Lgr5-GFP+ paneth cells; Lgr5-GFP- paneth cells
50μm

eeGGFFPP--TTRRFF11hhiigghh eexxpprreessssiioonn mmaarrkkss tthhee mmoorree pplluurriippootteenntt iiPPSS cceellllss
Dapi Nanog
25μm
eGFP-TRF1
Oct3/4-eGFP-TRF1
Nanog-eGFP-TRF1

eeGGFFPP--TTRRFF11hhiigghh iiPPSS cceellllss aarree mmoorree pplluurriippootteenntt ((tteerraattoommaass))
eGFP-TRF1+/+ low (40%); n=4
eGFP-TRF1+/+ high (40%); n=4
eGFP-TRF1+/KI low (40%); n=4
eGFP-TRF1+/+ unsorted; n=6
eGFP-TRF1+/KI unsorted; n=6
eGFP-TRF1KI/KI - unsorted; n=6
eGFP-TRF1+/KI- low (40%)
eGFP-TRF1+/KI- high (40%)
teratoma volume (mm3)
Day0 Day15 Day19 Day22 Day25 Day29
500
400
300
200
100
0
eGFP-TRF1+/KI high (40%); n=4
eGFP-TRF1+/KI high
Unsorted iPS
eGFP-TRF1+/+ low

Short telomeres as preditors ooff oonnsseett ooff hheerreeddiittaarryy bbrreeaasstt && oovvaarriiaann ccaanncceerr
Breast cancer: Familial breast & ovarian cancer
BRCA1/BRCA2
A C
BRCA1
Age (years)
Relative Telomere length
B D
BRCA2
Age (years)
3.5
3
2.5
2
1.5
1
0.5
3.5
3
2.5
2
1.5
1
0.5
0
-0.5
BRCAX
10 20 30 40 50 60 70 80
Age (years)
3.5
3
2.5
2
1.5
1
0.5
0
-0.5
Sporadic BC
10 20 30 40 50 60 70 80
Age (years)
0
-0.5
10 20 30 40 50 60 70 80
3.5
3
2.5
2
1.5
1
0.5
0
-0.5
10 20 30 40 50 60 70 80
Martínez-Delgado et al., PLoS Genetics, 2011
Martínez-Delgado et al., J. Medical Genetics, 2012 (ovarian)

HHaaiirr ffoolllliiccllee CCKK115--ppoossiittiivvee sstteemm cceellllss sshhooww tthhee hhiigghheesstt TTRRFF11--eeGGFFPP lleevveellss
3
2
1
0
6
4
2
avg. eGFP-TRF1 intensity
per area bg-reduced (a.u.)
nuclei:475
n=10 Inf.
p=0.004
n=12
n=12
CK15- CK15+
CK15
CK15 eGFP-TRF1 Dapi
eGFP-TRF1
CK15
eGFP-TRF1
Dapi
p=0.047
0
p= 0.015
Hair
bulb
Interfollicular
epidermis
Hair
bulge
Infundi-bulum
avg. eGFP-TRF1 intensity
per area bg-reduced (a.u.)
nuclei:299
n=11 bulges
nuclei:213
n=8 bulbs
nuclei:144
n=12 int. areas

A TERT based gene therapy of aging: ssiinnggllee ttrreeaattmmeenntt wwiitthh AAAAVV99--TTEERRTT
Lung
p= 0,03
n=3
AAV9-
mTERT
n=3
AAV9-
eGFP
p= 0,003
n=3
AAV9-
mTERT
n=3
AAV9-
eGFP
~ 1 yr ~2 yr
Fold changes in telomerase activity
AAV9-mTERT #1 AAV9-mTERT #2 AAV9-mTERT #3 AAV9-eGFP #1 AAV9-eGFP #2
P rRotneians (em g ) - + - 1 - 3 - 5+ 5 - 1 - 3 - 5+ 5 - -1 - 3 + 5 5 - - 1 - 3 + 5 -5 - 1- +3 5 5
Could be used in the treatment of some cases of
pulmonary fibrosis caused by telomerase mutations
Lung (2-year old group)
H
el
a
IC
3 3
3 months PI
Could be used in the treatment of some cases of
pulmonary fibrosis caused by telomerase mutations

eeGGFFPP--TTRRFF11hhiigghh iiPPSS cceellllss aarree mmoorree pplluurriippootteenntt ((cchhiimmeerraass))
D
0 to 30% chimerism
30 to 70% chimerism
70 to100% chimerism
57 embr.
2 pups
57 embr.
4 pups
61 embr.
2 pups
66 embr.
17 pups
low high low high
eGFP-TRF1+/+ eGFP-TRF1+/KI
10
8
6
4
2
0
(% of transfered embryos)
Chimeras
Generation of chimeras
iPS cells (C57BL/6J x agouti) with Hsd:ICR(CD-1) morulae
iPS clone Embryos
Injected
Cells
injected
Embryos
Transfered
Pups born Chimeras
iPS eGFP-TRF1+/+ pool cl. 2-8/1-5 LOW 57 5 to 6 57 2 1M (50%)
iPS eGFP-TRF1+/+ pool cl. 2-8/1-5 HIGH 57 5 to 6 57 4+ 1 dead 1M (40%) 1F (30%)
iPS eGFP-TRF1+/KI pool cl. 2-1/1-6 LOW 61 5 to 6 61 2+ 1dead 0
iPS eGFP-TRF1+/KI pool cl. 2-1/1-6 HIGH 66 5 to 6 66 17+ 3 dead 1M (80%) 1F (70%) 1M (50%)
1M (40%) 1F (30%)
iPS eGFP-TRF1+/+ iPS eGFP-TRF1+/KI
iPS eGFP-TRF1KI/KI

TRF1 is essential for bbootthh aadduulltt sstteemm cceellllss aanndd pplluurriippootteenntt sstteemm cceellllss
3 day old
TRF1D/D K5-Cre wildtype
1.14 g 2.53 g
TRF1D/D K5-Cre
hyperpigmentation
basal cell layer
hair
follicles
primitive hair
follicles
primitive hair
follicles
wildtype TRF1D/D K5-Cre
Lack of hair follicle
stem cells!

Telomere fragility Telomere fragility
80
60
40
20
0
Multitelomeric signals per metaphase
Wild type
Knock-out
Trf1 Tpp1 Rap1
……bbuutt ddooeess nnoott ccoorrrreellaattee wwiitthh ffrraaggiilliittyy
MTS: multitelomeric signals
(replication fork stalling at telomeres)

Mouse models to uunnddeerrssttaanndd tthhee rroollee ooff tteelloommeerraassee iinn ccaanncceerr && aaggiinngg
Blasco et al., Science (1995)
Blasco, Lee, et al., Cell (1997)
Lee, Blasco, et al., Nature (1998)
Telomerase inactivation models
Telomerase-deficient mice (Terc-/-)
Stem cell dysfunction
Decreased regenerative capacity
Diaseases associated with aging including
heart dysfuction
Less cancer
González-Suarez et al., Nat Genet (2000)
Flores et al., Science (2005)
Telomerase activation models
TERT transgenic mice (K5-TERT)
Less aging
Slightly more cancer
TERT Tg+ Sp53+Sp16+Sp19ARF (“triple”)
Delayed cancer, delayed aging & 40%
increase in lifespan
Wild-type “Triple”
González-Suarez et al., EMBO J. (2001)
Tomás-Loba et al, Cell (2008)

sshheelltteerriinn pprrootteeiinnss ((eexxcceepptt RRaapp11)) aarree eesssseennttiiaall ffoorr tteelloommeerree ccaappppiinngg
TTAGGG TTAGGG
AATCCC AATCCC
TTAGGG TTAGGG
AATCCC AATCCC
genes telomeres
genes telomeres
TTAGGG
TTAGGG
AATCCC AATCCC
TRF1
TRF2
cell death
cell senescence
aging
Pot1
TPP1
“shelterin”
Pot1
Tin2 TPP1
Rap1
TTPPPP11 rreeccrruuiittss tteelloommeerraassee ttoo cchhrroommoossoommee eennddss
TTRRFF11 iiss eennrriicchheedd iinn pplluurriippootteenntt sstteemm cceellllss aanndd iiss
eesssseennttiiaall ffoorr rreepprrooggrraammmmiinngg
TPP1
Rap1+/+ Rap1-/-

TRF1 is highly expressed in
adult stem cells and
pluripotent stem cells

Adult stem cells show tthhee hhiigghheesstt TTRRFF11--eeGGFFPP eexxpprreessssiioonn ((tteessttiiss))

TRF1 marks pluripotent
stem cells & is essential
for reprogramming

expre TRF1 is over-expresssseedd iinn EESSCCss aanndd iiPPSS cceellllss ccoommppaarreedd ttoo ssoommaattiicc cceellllss
TRF1

TRF1 is upregulated uuppoonn rreepprrooggrraammmmiinngg bbyy OOcctt33//44 pprreecceeeeddiinngg NNaannoogg
TRF1
MEF iPS 1 2 3 4 5 6 7 8 11 12
20
15
10
5
0
mRNA levels relative to MEFs
(set to 1)
MEF
(Days after 3F transduction)
N=2
Nanog
N=2
1 6 9 11 iPS
10
8
6
4
2
0
(relative to GAPDH x 10 -3 )
mRNA levels
MEF
(Days after 3F transduction)
p=0,5993
MEF iPS Oct3/4 Sox2 Klf4
15
10
5
0
p=0.0008
n=5
p=0.021
p=0,0421
p=0,0409p=0,0223 p=0,6174
n=5
n=5
n=5
n=5
p=0,7068
mRNA fold TRF1
(relative to MEF)
500
400
300
200
100
0
Ctrl Region Oct3/4 Pr TRF1 Pr Nanog Pr
p=0,001
n=4
p=0,272
p=0,002 p=0,097
n=4 n=4 n=4 n=4
n=4
n=4 n=3
n=4
n=4
n=4
n=3
p=0,969
p=0,005
p=0,539
p=0,007
mESC
ChIP-qRTPCR
No Antibody
OCT3/4
Nanog
Relative to Input x 10-3
Antibody
DNA levels
No Antibody
OCT3/4
Nanog
No Antibody
OCT3/4
Nanog
No Antibody
OCT3/4
Nanog
[ ...] Terf1
Ctrl Region TRF1 Pr
Nanog OCT3/4
OCT3/4
Nanog
Nanog Pr
Nanog OCT3/4
Oct3/4
OCT3/4 Pr

eeGGFFPP--TTRRFF11hhiigghh iiPPSS cceellllss eexxpprreessss mmoorree NNaannoogg,, TTEERRTT,, aanndd hhaavvee lloonnggeerr tteelloommeerreess
20
15
10
5
0
mRNA levels (relative to GAPDH)
eGFP-TRF1
p=0.02
n=3
n=3
p=0.04
n=3
n=3
150
100
low high low high MEF iPS
+/KI KI/KI +/+
2.
0
1.
5
1.
0
0.
5
0.
0
p=0.01
n=3
n=3
mTERT
p=0.04
n=3
n=3
+/KI KI/KI +/+
0
50
p=0.002
n=3
n=3
Nanog
p=0.001
n=3
n=3
+/KI KI/KI +/+
eGFP-TRF1+/KI
high low
Dapi; PNA-cy3
Tel-cy3 merge
10μm
high low wt r-cells
eGFP-TRF1+/KI
high low
eGFP-TRF1KI/KI
n=3 cl.
63 nuc.
3002 tel.
n=3 cl.
268 nuc.
10974 tel.
n=3cl.
64 nuc.
2514 tel.
n=1 cl.
46 nuc.
1996 tel.
n=3 cl.
56 nuc.
n=3cl. 4136tel.
69 nuc.
4511 tel.
eGFP-TRF1+/+
p=0.01
p=0.02
20
0
15
0
10
0
50
0
mRNA levels (relative to GAPDH)

TTRRFF11--nnuullll MMEEFFss ccaannnnoott bbee rreepprrooggrraammmmeedd…….. eevveenn iinn tthhee aabbsseennccee ooff pp533
TRF1+/+p53-/- -Cre TRF1lox/loxp53-/- -Cre
200.000 cells
1FRT xol/ xolFEM
1FRFTEM eΔr/ΔC-
1.5Kb (TRF1lox)®
0.48 Kb (TRF1Δ) ®
TRF1lox/loxp53-/-- Cre iPS clones
1 2 3 4 5 6 7
1,2 n=6
1
0,8
0,6
0,4
0,2
TRF1+/+
p53-/-- Cre
p<0.0001
TRF1Δ/Δ
p53-/-- Cre
TRF1lox/lox
p53-/-- Cre
Relative reprogramming efficiency
0
n=4
n=5
p=0.0008
(locus excised
by PCR)
12
10
8
6
4
2
0
2 5 6
Day post-infection
Positive cells for SSEA-1 (%)
Not reprogrammed MEFs
TRF1+/+p53-/-- Cre, n=3
TRF1lox/loxp53-/-- Cre, n=3

TTRRFF11 iiss eesssseennttiiaall ffoorr mmaaiinntteennaannccee ooff pplluurriippootteennccyy
Day 0
TRF1+/+
iPS
TRF1lox/lox
iPS
-4-OHT
+4-OHT
Day 6 Day 9
120
100
80
60
40
20
0
TRF1+/+ iPS
+4-OHT
(day10)
TRF1lox/lox
iPS +4-OHT
(day10)
Colonies with cell death
Differentiated colonies
Normal colonies
1,
4
1,
2
1
0,
8
0,
6
0,
4
0,
2
0
TRF1+/+ iPS + 4-OHT
TRF1lox/lox iPS+ 4-OHT
n=5
n=5

telo The importance of telommeerree ccaappppiinngg ffoorr iiPPSS cceellll ggeenneerraattiioonn
Failure to reprogram
Oct3/4
parental cells iPS cells
Tejera et al., Developmental Cell, 2010
Marión et al., Cell Stem Cell, 2009
Marion et al., Nature, 2009
TRF1-/-
3F
WT
TRF1
NanogTERT
Tpp1+/+ Net telomere elongation

Identification ooff ccoommppoouunnddss tthhaatt ddiissrruupptt TTRRFF11 bbiinnddiinngg

ETP-00047363 ETP-00047228 8h
24h
Het
C DMSO
Het
C DMSO
Het
C DMSO
ETP-00047361
ETP-00041108
Het
C DMSO
Het
C DMSO
ETP-00047037
Mean fluorescence intensity
(Normalized to Control DMSO)
Mean fluorescence intensity
(Normalized to Control DMSO)
IIddeennttiiffiiccaattiioonn ooff aa ttoottaall ooff 2200--hhiittss

Delayed aaggiinngg && lliiffee--eexxtteennssiioonn bbyy TTEERRTT rreeqquuiirreess iittss ccaattaallyyttiicc aaccttiivviittyy
Survival
Percent 0 55
Weeks 100
50
0
AAV9-eGFP n=12
80 120 160
V. I.
24%
AAV9-mTERT n=21
AAV9-mTERT-DN n=17
13%
eGFP vs mTERT
p= 0,02 (Log Rank)
mTERT-DN vs mTERT
p= 0,03 (Log Rank)
eGFP vs mTERT-DN
p= 0,52 (Log Rank)

rem Increased remooddeelliinngg ppoosstt MMII aassssoocciiaatteedd ttoo TTEERRTT eexxpprreessssiioonn
ECM
rel. expression
I: infarct
IT: infarct + Tert treatment
AAV9
empty
AAV9
Tert
rel. expression
down up
FDR<10e-04
ECM
I IT
p=0.047
n=9
FGFR
n=9
FDR=0.075
FGFR
2.5
2.0
1.5
1.0
0.5
I IT
AAV9
empty
TGFß
AAV9
Tert
TGFß
rel. expression
down up
Fgfr3
2.5
2.0
1.5
1.0
0.5
0.0
p=0.002
n=6
n=6
AAV9-empty
AAV9-Tert
AAV9
empty
AAV9
Tert
down up
FDR=0.085
I IT
0.0
Bmprb1

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