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Targeting Telomeres in Human Disease:Advances and Therapeutic OpportunitiesTargeting Telomeres in Human Disease:

Advances and Therapeutic Opportunities


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Targeting

Telomeres

in

Cancer

dvances

and

Therapeutic

Opportunities

RogerReddelMBBSPhD

Children'sMedicalResearchInstitute

UniversityofSydney


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Telomerestructure

Centromere (5'TTAGGG3')n

(3'AATCCC5')n

DNAsequence


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Telomerestructure


(3'AATCCC5')n

DNAsequence

Shelterin proteins


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Telomerestructure


(3'AATCCC5')n

DNAsequence

Tloopformation

Shelterin proteins


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Telomerestructure


(3'AATCCC5')n

Gquadruplex

G

G

G

G G

G

G G

G G G

G G

Gquartet

G

G

G

G


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Telomeresshortenwithreplication

Telomeresshortenevery

timecellsdivide

STOP


(3'AATCCC5')n

DDR


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Telomerelengtheningprocesses


(3'AATCCC5')n TelomeraseTERT

Dyskerin


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(5'TTAGGG3')n


Dyskerin

AlternativeLengthening

ofTelomeres


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(5'TTAGGG3')n


Dyskerin


ofTelomeres


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(5'TTAGGG3')n


Dyskerin


ofTelomeres


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(5'TTAGGG3')n


Dyskerin


ofTelomeres


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(5'TTAGGG3')n


Dyskerin


ofTelomeres


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(5'TTAGGG3')n


Dyskerin


ofTelomeres


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Rapidtelomereshortening Telomeretrimming

Overlengthening


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Rapidtelomereshortening Telomeretrimming

Shortenedtelomere

Tcircle

Overlengthening

Telomererefolded

Pickett,HAetal.EMBOJ(2009)


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TRAPassay

TERT

Dyskerin

substrate

TERT

Dyskerin

dTTP

dATP

dGTP

substrate

extended

PCR

radiolabel

KimNWetal.,Science(1994)

Detectingtelomeraseintumors

ALT

TEL


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IPTRAPassay

TERT

Dyskerin

TERT

Dyskerin

peptide

antigen

TERT

Dyskerin

substrate

TERT

Dyskerin

dTTP

dATP

dGTP

PCRradiolabel

Au,AYetal.,LungCancer(2011)


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Directtelomeraseassay

TERT

Dyskerin

TERT

Dyskerin

TERT

Dyskerin

TERT

Dyskerin

peptide

antigen

dTTP

dATP

dGTP

radiolabel

substratesubstrate

extended

CohenSBetal.,NatMethods(2008)


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Detecting LTintumors

TerminalRestrictionFragmentSouthernblot

=restriction

enzymes

that

do

not

recognise

TTAGGG

Gelelectrophoresistoseparatebyfragmentsize

Southernblot,denature

Radiolabeledtelomeric probe

23

9.4

6.6

4.3

kb

ALT

TEL


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ALTassociatedPMLbodies

JiangWQetal.,inK.Hiyama ed."Telomeresand

TelomeraseinCancer",HumanaPress


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CCircleassay

CCircle 29

polymerase

Dotblotxxx

Radiolabeledprobe

Rolling

circle

amplification

HensonJDetal.,NatBiotechnol (2009)


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Tumors thatuse LT

Predominant

Osteosarcomas

Undifferentiatedpleomorphicsarcomas

LeiomyosarcomasGrade2&3astrocytic braintumors

Common

LiposarcomasGlioblastoma multiforme

Neuroblastoma

Rare

Rhabdomyosarcoma

Mosttypesofcarcinomas

HensonJDetal.,FEBSLett (2010)


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Prognosticsignificanceoftelomeraseand LT

CorrelationbetweenprobabilityofsurvivalandTMMdependsontumortype

Glioblastoma multiforme

ALT>>TEL

Osteosarcoma

None>TELorALT

Liposarcoma

None>TEL>ALT

Ulaner,GAetal.,CancerRes.(2003)

Costa,Aetal.,CancerRes.(2006)

HakinSmith,Vetal.,Lancet(2003)


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Targeting

telomeres

in

cancer potential

opportunities

InhibitingTelomeraseactivity

TERT

Dyskerin

TERT

Dyskerin

Biogenesis Transport

DockingCatalysis

Transportto

anothertelomere


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Targetingtelomeresincancer potentialopportunities

InhibitingALTactivity

Juxtapositionoftelomeres


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Telomeremaintenancespecificsurfaceantigens

TERT

Dyskerin


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Canabnormaltelomerearchitecturebetargetedincancercellswithupregulated

telomeraseorALT?

ALT:variantDNAsequences

ALT:decreasedshelterin binding

Conomos,Detal.,JCellBiol.(2012)

Cesare,AJetal.,NatStr Mol Biol.(2009)

Dejardin,Jetal.Cell(2009)


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ArecancercellswithupregulatedtelomeremaintenancemorevulnerabletoG4ligands?

G

G

G G

G G G

G G


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Gainorlossoftransactingfactors

TA

TERTpromoter

Prodrugactivator


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Gainorlossoftransactingfactors

ALT

Repressor RepressionofALT

Treatment

Synthetic

lethality?


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Telomeretrimming

Shortenedtelomere

Tcircle

Canwestimulatetelomeretrimmingselectivelyincancercells?


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Targetingtelomeresincancer challenges

1. Lengthoftimetoact


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Kb

48.5

19.4

8.6

2.8

90 10028 48 58 68 82 11038 120

PopulationDoublings(PD)

JiangWQetal.,Mol CellBiol (2005)


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2. Potentialsideeffectsonnormalcellsthatrequiresometelomeremaintenance,

andonthegermline

3. Developmentofresistance: ALT telomerase

4. CancercellsthathavenoTMM


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JudithHyam Memorial

TrustFund



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Sponsored by:

ParticipatingExperts:

Brought to you by the Science/AAAS Custom Publishing Office


30 Apri l 2014

Roger Reddel, M.B., B.S., Ph.D.

Children's Medical Research Institute

Sydney, Australia

Suneet Agarwal, M.D., Ph.D.

Boston Children's Hospital

Boston, MA

Targeting Telomeres in Human Disease:Advances and Therapeutic OpportunitiesTargeting Telomeres in Human Disease:Advances and Therapeutic Opportunities


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Dyskeratosis congenita:the prototypic telomere disease



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Dyskeratosis congenita (DC)

Zinsser-Cole-Engman syndrome,

first described in early 1900s

Incidence: 1/1,000,000?

Major manifestations (Costello andBunke, 1956):

Skin pigmentation changes

nail abnormalities white plaques on mucosal surfaces

Major causes of illness and death

Aplastic anemia

Lung disease

Cancer: blood and skin/epithelial

Liver failure

Median overall survival: 42 years Walne and Dokal, 2008

Alter, et al., 2009


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Dyskeratosis congenita genetic discovery

1980s - linkage analysis in X-linked patients: Xq28 1995 DC Registry established at Hammersmith Hospital, UK;

candidate screening in refined 1.4 cM region of Xq28

(Dokal, Vulliamy and colleagues)

DKC1: homolog of yeast Cbf5, a pseudouridine synthase Binds box H/ACA small nucleolar RNAs

Role in rRNA modifications, ribosomal biogenesis?

Southern blot of DKC1 locus

Heiss et al,

Nature Genetics, 1998

5 DC patients with

missense mutations


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Dyskeratosis congenita genetic discovery

dyskerin interacts with human telomerase RNA (TERC) viaa box H/ACA motif (Mitchell and Collins, Nature, 1999)

TERC

U64

Northern blot for TERC in DKC1 patient samples

TERC

Box H/ACA

Nature 2001

TERC mutations in a DC family

D k i i


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Dyskeratosis congenita-

a disorder of telomere maintenance

-Telomeres protect chromosome ends

-Telomere length is associated with cellular replicative capacity

-Telomerase extends telomeres

Calado and Young

Blood 2008


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Telomere length

Southern blot

DC patient

normal

Populationdoub

lings Fibroblast growth

DC M F

Westin, et al

Aging Cell 2007

Telom

erelength

Telomere length by passagenormal DC patient

10 21 60 10 33 passage

Telomere length and replicative capacity

are impaired in DC patient cells


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Measuring telomere length flow-FISH

Quantitative fluorescence in situ hybridization / flow cytometry

TTAGGGTTAGGGTTAGGG

Lansdorp and colleagues

- Telomere length 90% sensitivity, > 85% positive predictive value

- in granulocytes 96% sensitivity, but only 69% positive predictive value

Alter et al, Blood 2007

CCCTAACCCTAACCCTAA

DC

Non DC

T l t ti i DC 2014


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Telomere gene mutations in DC - 2014

Telomerase: DKC1, TERC, TERT, TCAB1, NOP10, NHP2 Telomere components / replication machinery: TINF2, CTC1, RTEL1

X-linked, autosomal dominant, autosomal recessive, sporadic

40% still unknown

TERT

TERC

DKC1

TINF2

Calado and Young

Blood 2008

Red: dyskeratosis congenita

Green: idiopathic aplastic anemiaBlack: pulmonary fibrosis

A t f t l di


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A spectrum of telomere diseases

Bessler, et al

FEBS, 2010

-DKC1 and TINF2: severe infantile-onset disorders

Revesz and Hoyeraal Hreidarsson syndromes

-TERT and TERC: later-onset

Idiopathic aplastic anemia/MDS, idiopathic pulmonary fibrosis

G ti ti i ti i t l di


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Genetic anticipation in telomere diseases

Autosomal

Dominant DC

Vulliamy et al,

Nature Genetics, 2004

Telome

relength

Telomere repeats

Initial length in parent with AD-DC

Initial length in his/her child

Size of functionalproblems


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Pleiotropy and


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15 year old with bone

marrow failure

Gray hair

Oral plaques

Skin pigment changes

Cracked nails

Very short telomere length

Negative for mutations inknown genes

Pleiotropy and

phenotypic variation

MA01-104


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Brain cysts Bone lesions

Eye vessel

abnormalities

Gut vessel

abnormalities

Anderson, et al., Nature Genetics, 2012

- exome analysis in Coats plus patients reveals CTC1 mutations

Coats plus link to DC?


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Coats plus link to DC?

Some patients have sparse, gray hair; nail changes; low blood counts

CTC1: part of a conserved complex involved in telomere maintenance

Hypothesis: some individuals with DC may have mutations in CTC1

CTC1 mutations in a DC patient


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Keller, et al,

Pediatric Blood and Cancer, 2012

CTC1 mutations in a DC patient

c.2954_2956delGTT

CTC1 alleles in DC:


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CTC1 alleles in DC:

identical to those in Coats plus families

Anderson, et al.,

Nature Genetics 2012

Phenotypic overlap with Coats plus syndrome


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Phenotypic overlap with Coats plus syndrome

Thalamic calcification Large syrinx

Keller, et al, PBC, 2012

GI: tiny vascular lesionsObliterated peripheral retinal

vessels


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CTC1/Coats plus extends the spectrum of telomere

biology disorders

SavageNature Genetics, 2012

-6 of 73 patients in UK DC Registry harbored compound

heterozygous CTC1 mutations (Walne, et al., Haematologica, 2012)

Somatic reversion in autosomal dominant DC


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TERC gene

So a c e e s o au oso a do a C

Jongmans, et al, AJHG, 2012

-seven different events of reversion of TERC mutation caused by somaticrecombination in blood cells of six patients in four DC families

-uniparental disomy in both myeloid and lymphoid cells, indicating event in

early hematopoietic progenitor

-in vivo selective advantage of hematopoietic progenitors with twofunctional copies of TERC

Summary: insights from genetic discovery in


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Summary: insights from genetic discovery in

DC / telomere diseasesDiagnosis is aided by:

- Functional testing: age-adjusted mean telomere length

- Genes: DKC1, TERC, TERT, TINF2, NOP10, NHP2, TCAB1,CTC1, RTEL1,...

Confounding factors:

-Incomplete genetic characterization

-Multiple modes of inheritance

-Genetic anticipation

-Variable penetrance, pleiotropy and the telotype

-Somatic reversion

- evaluate patients of any age presenting with aplastic anemia,

associated signs, or suspicious family history, for DC/telomere disease

- Implications for management, therapy, family counseling

Bone marrow transplantation for DC


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Bone marrow transplantation for DC

Allogeneic BMT is curative for the blood defects in DC

Poor outcomes in DC patients undergoing conventional

BMT, due to increased early and late complications

Conventional allogeneic BMT


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Conventional allogeneic BMT

Blood stem cells

2. Transplant

-anti-tumor

-immune suppression

-create space

-collateral tissue damage

-short and long-term sequelae

-graft-versus-tumor effect

-cell or enzyme replacement

-graft-versus-host disease

-immunosuppression: toxicity and risks

1. Conditioning

Chemotherapy

Alkylating agents

Radiation

Outcome after BMT in DC


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Alter, et al. Blood 2009: reviewed 65 cases in literature

Cum

ulativesurviv

al

Sibling donor

Alternate donor

Long term survival < 25%

predisposition to pulmonary, hepatic, vascular complications and

secondary malignancy

conventional conditioning regimens: radiation and alkylators

Trial: Radiation and alkylator free


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Rationale/hypotheses:

Non-malignant disease:

as long as engraftment is not compromised, decreasing BMTconditioning toxicity will improve outcomes

eliminating alkylator and radiation exposure will decrease organtoxicity (e.g. liver, lung) and cancer

The telomere defect in DC results in a replicative disadvantage inhematopoietic and immune cells, which will favor engraftment

Trial: Radiation- and alkylator-free

BMT for bone marrow failure in DC patients

Radiation and alkylator free BMT for DC?


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Radiation and alkylator-free BMT for DC?

Blood stem cells

2. Transplant

Immune

Suppression? engraftment

1. Conditioning

Alkylating agents

Radiation

X

X

Radiation- and alkylator-free BMT in DC patients


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Conditioning

Graft versus hostdisease prophylaxis

http://clinicaltrials.gov/ct2/show/NCT01659606

y p

CAM: Campath-1H: anti-CD52 antibody

FLU: fludarabine: purine analog

opened July 2012 4 DC patients treated; all engrafted

Summary


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y

Dyskeratosis congenita

a telomere biology disorder, affecting self-

renewal of cells in various tissues

Genetics and disease manifestations are

complex

Diagnostic and management challenges

Applying new knowledge in disease-specific

clinical trials

Acknowledgements


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BCH Hematology/Oncology

Leslie Lehmann

George Daley

Inga Hofmann

Sung-Yun Pai

David Williams

Acknowledgements

Clinical and Translational

Investigation Program

Wendy London

Sarah HuntJane OBrien

Agarwal lab

Rayhnuma Ahmed

Katelyn GagneRachel Keller

Diane Moon

Matthew Segal

Funding

Harvard Stem Cell Institute

Charles H. Hood FoundationAmerican Society of Hematology



68/69

Sponsored by:

ParticipatingExperts:

Brought to you by the Science/AAAS Custom Publishing Office

To submit your questions,type them into the text box

and click .

30 Apri l 2014

Targeting Telomeres in Human Disease:Advances and Therapeutic OpportunitiesTargeting Telomeres in Human Disease:Advances and Therapeutic Opportunities

Roger Reddel, M.B., B.S., Ph.D.

Children's Medical Research Institute

Sydney, Australia


Boston Children's Hospital

Boston, MA


T ti T l i H DiT ti T l i H Di


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Targeting Telomeres in Human Disease:Advances and Therapeutic Opportunities

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