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Webinar SeriesWebinar SeriesScienceScience

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

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Participating Experts:

Brought to you by the Science/AAAS Custom Publishing Office


30 April 2014

Roger Reddel, M.B., B.S., Ph.D.Children's Medical Research InstituteSydney, Australia

Suneet Agarwal, M.D., Ph.D.Boston Children's HospitalBoston, MA


Targeting Telomeres in Cancer: Advances and Therapeutic Opportunities

Roger Reddel MBBS PhD

Children's Medical Research Institute

University of Sydney

Telomere structure

Centromere (5'‐TTAGGG‐3')n

(3'‐AATCCC‐5')nDNA sequence

Telomere structure



Shelterin proteins

Telomere structure



T‐loop formation

Shelterin proteins

Telomere structure


(3'‐AATCCC‐5')n

G‐quadruplex

GG

G

G G

G

G G

G G G

G G

G‐quartet

G

G

G

G

Telomeres shorten with replication

Telomeres shorten every time cells divide

STOP


(3'‐AATCCC‐5')n

DDR

DDR = DNA damage response

Telomere lengthening processes


(3'‐AATCCC‐5')n TelomeraseTERT

Dyskerin

Telomere lengthening processes

(5'‐TTAGGG‐3')n

(3'‐AATCCC‐5')n TelomeraseTERT

Dyskerin

Alternative Lengtheningof Telomeres

Rapid telomere shortening: Telomere trimming

Over‐lengthening


Over‐lengthening

Shortened telomere

T‐circle


Shortened telomere

T‐circle

Over‐lengthening

Telomere re‐folded

Pickett, HA et al. EMBO J (2009)

TRAP assay

TERT

Dyskerin

substrate

TERT

Dyskerin

dTTPdATPdGTP

substrateextended

PCRradiolabel

Kim NW et al., Science (1994)

Detecting telomerase in tumors

ALT

TEL


IP‐TRAP assay

TERT

Dyskerin

TERT

Dyskerin

peptide antigen

TERT

Dyskerin

substrate

TERT

Dyskerin

dTTPdATPdGTP

PCRradiolabel

Au, AY et al., Lung Cancer (2011)


Direct telomerase assay

TERT

Dyskerin

TERT

Dyskerin

TERT

Dyskerin

TERT

Dyskerin

peptide antigen

dTTPdATPdGTP

radiolabel

substratesubstrateextended

Cohen SB & Reddel RR, Nat Methods (2008)

Detecting ALT in tumors

Terminal Restriction Fragment Southern blot

= restriction enzymes that do not recognise TTAGGG

Gel electrophoresis to separate by fragment size

Southern blot

Radiolabeled telomeric probe

23

9.4

6.64.3

kb

ALT

TEL


ALT‐associated PML bodies

Jiang WQ et al., in K. Hiyama ed. "Telomeres and Telomerase in Cancer", Humana Press


C‐Circle assay

C‐Circle Φ29 polymerase

Dot blot xxxRadiolabeled probe

Rollingcircle

amplification

Henson JD et al., Nat Biotechnol (2009)

Tumors that use ALT

Predominant

OsteosarcomasUndifferentiated pleomorphic sarcomas

LeiomyosarcomasGrade 2&3 astrocytic brain tumors

Common

LiposarcomasGlioblastoma multiforme

Neuroblastoma

Uncommon

RhabdomyosarcomaMost types of carcinomas

Henson JD et al., FEBS Lett (2010)

Prognostic significance of telomerase and ALT

Correlation between probability of survival and TLM depends on tumor type

Glioblastoma multiforme

ALT >> TEL

Osteosarcoma

None > TEL or ALT

Liposarcoma

None > TEL > ALT

Ulaner, GA et al., Cancer Res. (2003)Costa, A et al., Cancer Res. (2006)Hakin‐Smith, V et al., Lancet (2003)

TLM = telomere lengthening mechanismTEL = telomerase

Targeting telomeres in cancer: potential opportunities

Inhibiting Telomerase activity

TERT

Dyskerin

TERT

Dyskerin

BiogenesisTransport

DockingCatalysis

Transport to another telomere


Inhibiting ALT activity

Juxtaposition of telomeres


Telomere maintenance‐specific surface antigens

TERT

Dyskerin


Can abnormal telomere architecture be targeted in cancer cells with up‐regulated telomerase or ALT?

ALT: variant DNA sequences

ALT: decreased shelterin binding

Conomos, D et al., J Cell Biol. (2012)Cesare, AJ et al., Nat Str Mol Biol. (2009)Dejardin, J et al. Cell (2009)


Are cancer cells with up‐regulated telomere maintenance more vulnerable to G4 ligands?

G

G

G G

G G G

G G


Gain or loss of trans‐acting factors

TA

TERT promoter

Pro‐drug activator


Gain or loss of trans‐acting factors

ALT Repressor Repression of ALT

Treatment

Syntheticlethality?


Telomere trimming

Shortened telomereT‐circle

Can we stimulate telomere trimming selectively in cancer cells?

Targeting telomeres in cancer: challenges1. Length of time to act


Kb

48.5

19.4

8.6

2.8

90 10028 48 58 68 82 11038 120

Population Doublings (PD)

Jiang WQ et al., Mol Cell Biol (2005)


2. Potential side‐effects on normal cells that require some telomere lengthening,and on the germ‐line

3. Development of resistance: ALT telomerase

4. Cancer cells that have no telomere lengthening mechanism

Judith Hyam Memorial Trust Fund

Sponsored by:




30 April 2014




Dyskeratosis congenita:the prototypic telomere disease

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

Dyskeratosis congenita (DC)• Zinsser-Cole-Engman syndrome,

first described in early 1900s

• Incidence: 1/1,000,000?

• Major manifestations (Costello and Bunke, 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

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

Dyskeratosis congenita – genetic discovery• dyskerin interacts with human telomerase RNA (TERC) via

a 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

Dyskeratosis congenita-a disorder of telomere maintenance

-Telomeres protect chromosome ends-Telomere length is associated with cellular replicative capacity-Telomerase extends telomeres

Calado and YoungBlood 2008

Telomere length Southern blot

DC patient

normal

Pop

ulat

ion

doub

lings

Fibroblast growthDC M F

Westin, et alAging Cell 2007

Telo

mer

e le

ngth

Telomere length by passagenormal DC patient

10 21 60 10 33 passage

Telomere length and replicative capacityare impaired in DC patient cells

Measuring telomere length – flow-FISH• Quantitative fluorescence in situ hybridization / flow cytometry

TTAGGGTTAGGGTTAGGG

Lansdorp and colleagues

- Telomere length <1%ile:- in lymphocytes >90% sensitivity, > 85% positive predictive value- in granulocytes 96% sensitivity, but only 69% positive predictive value

Alter et al, Blood 2007

CCCTAACCCTAACCCTAA

DCNon DC

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 YoungBlood 2008

Red: dyskeratosis congenitaGreen: idiopathic aplastic anemiaBlack: pulmonary fibrosis

A spectrum of telomere diseases

Bessler, et alFEBS, 2010

-DKC1 and TINF2: severe infantile-onset disorders Revesz and Hoyeraal Hreidarsson syndromes

-TERT and TERC: later-onsetIdiopathic aplastic anemia/MDS, idiopathic pulmonary fibrosis

Genetic anticipation in telomere diseases

Autosomal Dominant DC

Vulliamy et al, Nature Genetics, 2004

Telo

mer

e le

ngth

Telomere repeats

Initial length in parent with AD-DC

Initial length in his/her child

Size of functional problems

Variable penetrance and the “telotype”

TERTTERTA716V

BloodLiverLung

Diaz de Leon et alPLOS One, 2010

Telomere length

TERT mutation: Y N Y N Y

• 15 year old with bone marrow failure

• Gray hair• Oral plaques• Skin pigment changes• Cracked nails• Very short telomere length• Negative for mutations in

known genes

Pleiotropy andphenotypic variation

MA01-104

Brain cysts Bone lesions

Eye vesselabnormalities

Gut vesselabnormalities

Anderson, et al., Nature Genetics, 2012

- exome analysis in Coats plus patients reveals CTC1 mutations

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

Keller, et al, Pediatric Blood and Cancer, 2012

CTC1 mutations in a DC patient

c.2954_2956delGTT

CTC1 alleles in DC:identical to those in Coats plus families

Anderson, et al., Nature Genetics 2012

Phenotypic overlap with Coats plus syndrome

Thalamic calcification Large syrinx

Keller, et al, PBC, 2012GI: tiny vascular lesionsObliterated peripheral retinal

vessels

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)

TERC gene

Somatic reversion in autosomal dominant DC

Jongmans, et al, AJHG, 2012

-seven different events of reversion of TERC mutation caused by somatic recombination 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 two functional copies of TERC

Summary: insights from genetic discovery in DC / telomere diseases

Diagnosis 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

• 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

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

ChemotherapyAlkylating agents

Radiation

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

Outcome after BMT in DC

Cum

ulat

ive

surv

ival

Sibling donorAlternate donor

• Long term survival < 25%• predisposition to pulmonary, hepatic, vascular complications and secondary malignancy• conventional conditioning regimens: radiation and alkylators

Rationale/hypotheses:

– Non-malignant disease: as long as engraftment is not compromised, decreasing BMT conditioning toxicity will improve outcomes

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

– The telomere defect in DC results in a replicative disadvantage in hematopoietic 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?

Blood stem cells

2. Transplant

ImmuneSuppression ? engraftment

1. Conditioning

Alkylating agents

Radiation

XX

Conditioning

Graft versus host disease prophylaxis

http://clinicaltrials.gov/ct2/show/NCT01659606Radiation- and alkylator-free BMT in DC patients

• CAM: Campath-1H: anti-CD52 antibody• FLU: fludarabine: purine analog

• opened July 2012• 4 DC patients treated; all engrafted

Summary

• Dyskeratosis congenitaa 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

BCH Hematology/OncologyLeslie Lehmann George DaleyInga HofmannSung-Yun PaiDavid Williams

AcknowledgementsClinical and Translational Investigation ProgramWendy LondonSarah HuntJane O’Brien

Agarwal labRayhnuma Ahmed Katelyn GagneRachel KellerDiane MoonMatthew Segal

FundingHarvard Stem Cell InstituteCharles H. Hood FoundationAmerican Society of Hematology

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