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Webinar SeriesWebinar SeriesScienceScience
Targeting Telomeres in Human Disease:Advances and Therapeutic OpportunitiesTargeting Telomeres in Human Disease:Advances and Therapeutic Opportunities
Sponsored by:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
Webinar SeriesWebinar SeriesScienceScience
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 Human Disease:Advances and Therapeutic OpportunitiesTargeting Telomeres in Human Disease:Advances and Therapeutic Opportunities
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
Centromere (5'‐TTAGGG‐3')n
(3'‐AATCCC‐5')nDNA sequence
Shelterin proteins
Telomere structure
Centromere (5'‐TTAGGG‐3')n
(3'‐AATCCC‐5')nDNA sequence
T‐loop formation
Shelterin proteins
Telomere structure
Centromere (5'‐TTAGGG‐3')n
(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
Centromere (5'‐TTAGGG‐3')n
(3'‐AATCCC‐5')n
DDR
DDR = DNA damage response
Telomere lengthening processes
Centromere (5'‐TTAGGG‐3')n
(3'‐AATCCC‐5')n TelomeraseTERT
Dyskerin
Telomere lengthening processes
(5'‐TTAGGG‐3')n
(3'‐AATCCC‐5')n TelomeraseTERT
Dyskerin
Alternative Lengtheningof Telomeres
Telomere lengthening processes
(5'‐TTAGGG‐3')n
(3'‐AATCCC‐5')n TelomeraseTERT
Dyskerin
Alternative Lengtheningof Telomeres
Telomere lengthening processes
(5'‐TTAGGG‐3')n
(3'‐AATCCC‐5')n TelomeraseTERT
Dyskerin
Alternative Lengtheningof Telomeres
Telomere lengthening processes
(5'‐TTAGGG‐3')n
(3'‐AATCCC‐5')n TelomeraseTERT
Dyskerin
Alternative Lengtheningof Telomeres
Telomere lengthening processes
(5'‐TTAGGG‐3')n
(3'‐AATCCC‐5')n TelomeraseTERT
Dyskerin
Alternative Lengtheningof Telomeres
Telomere lengthening processes
(5'‐TTAGGG‐3')n
(3'‐AATCCC‐5')n TelomeraseTERT
Dyskerin
Alternative Lengtheningof Telomeres
Rapid telomere shortening: Telomere trimming
Over‐lengthening
Rapid telomere shortening: Telomere trimming
Over‐lengthening
Shortened telomere
T‐circle
Rapid telomere shortening: Telomere trimming
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
Detecting telomerase in tumors
IP‐TRAP assay
TERT
Dyskerin
TERT
Dyskerin
peptide antigen
TERT
Dyskerin
substrate
TERT
Dyskerin
dTTPdATPdGTP
PCRradiolabel
Au, AY et al., Lung Cancer (2011)
Detecting telomerase in tumors
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
Detecting ALT in tumors
ALT‐associated PML bodies
Jiang WQ et al., in K. Hiyama ed. "Telomeres and Telomerase in Cancer", Humana Press
Detecting ALT in tumors
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
Targeting telomeres in cancer: potential opportunities
Inhibiting ALT activity
Juxtaposition of telomeres
Targeting telomeres in cancer: potential opportunities
Telomere maintenance‐specific surface antigens
TERT
Dyskerin
Targeting telomeres in cancer: potential opportunities
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)
Targeting telomeres in cancer: potential opportunities
Are cancer cells with up‐regulated telomere maintenance more vulnerable to G4 ligands?
G
G
G G
G G G
G G
Targeting telomeres in cancer: potential opportunities
Gain or loss of trans‐acting factors
TA
TERT promoter
Pro‐drug activator
Targeting telomeres in cancer: potential opportunities
Gain or loss of trans‐acting factors
ALT Repressor Repression of ALT
Treatment
Syntheticlethality?
Targeting telomeres in cancer: potential opportunities
Telomere trimming
Shortened telomereT‐circle
Can we stimulate telomere trimming selectively in cancer cells?
Targeting telomeres in cancer: challenges1. Length of time to act
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)
Targeting telomeres in cancer: challenges1. Length of time to act
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:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
Webinar SeriesWebinar SeriesScienceScience
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 Human Disease:Advances and Therapeutic OpportunitiesTargeting Telomeres in Human Disease:Advances and Therapeutic Opportunities
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
Sponsored by:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
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Webinar SeriesWebinar SeriesScienceScience
30 April 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 InstituteSydney, Australia
Suneet Agarwal, M.D., Ph.D.Boston Children's HospitalBoston, MA
Brought to you by the Science/AAAS Custom Publishing OfficeBrought to you by the Science/AAAS Custom Publishing Office
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Sponsored by:
Webinar SeriesWebinar SeriesScienceScience
30 April 2014
Targeting Telomeres in Human Disease:Advances and Therapeutic OpportunitiesTargeting Telomeres in Human Disease:Advances and Therapeutic Opportunities