The preclinical efficacy of a novel telomerase inhibitor, imetelstat ... · Baerlocher et al., NEJM...

The preclinical efficacy of a novel telomerase inhibitor, imetelstat, in AML: A randomized trial in

patient-derived xenografts

Claudia Bruedigam, Ph.D Gordon and Jessie Gilmour Leukaemia Research Laboratory Headed by A/Professor Steven Lane

Telomerase is activated to maintain the long-term replicative potential in most cancers including AML

© QIMR Berghofer Medical Research Institute | 2

•  Telomerase is overexpressed in most AML

Roth et al., Leukemia 2003

•  AML oncogenes activate telomerase Gessner et al., Leukemia 2010

•  LSC have shortened telomeres and increased telomerase activity Drummond et al., Leukemia 2005, Bernard et al., Leukemia 2009

•  Genetic depletion of telomerase eradicates LSC upon enforced replication via cell cycle arrest and apoptosis

Bruedigam et al., Cell Stem Cell 2014

Bruedigam et al., Cell Stem Cell 2014-

Calado and Young, N Engl J Med 2009

Imetelstat (JNJ-63935937) is a competitive inhibitor of telomerase activity


•  Imetelstat is a covalently lipidated 13-mer oligonucleotide that binds the RNA template of telomerase

Herbert et al., Oncogene 2005

•  Imetelstat induced molecular and

complete hematological responses in essential thrombocythemia (89%)

Baerlocher et al., NEJM 2015

•  Imetelstat showed efficacy in myelofibrosis (complete or partial remission in 21%)

Tefferi et al., NEJM 2015

•  Phase II / III trial to evaluate imetelstat in low or intermediate-1 risk myelodysplastic syndrome

NCT02598661

Ruden et al, Cancer Treatment Reviews 2013


Generating an AML patient-derived xenograft inventory

Sublethal irradiation(2.8 Gy)

NSGS

Primary AML patient sample

Ficoll separation

CD3 depletion

EngraftmentAML symptoms

Engraftment(Peripheral blood donor chimerism)

Weeks post-transplant

Dono

r chi

mer

ism

[%]

0 10 20 30 40 500

20

40

60

80

100engraftednot engraftedongoing

Blast morphology(Wright-Giemsa: peripheral blood)

Survival

0 50 100 150 200 250 300 3500

50

100

Perc

ent s

urvi

val

Days post-transplant

Individual samples tested (#): 35

Median survival (days): 220Engraftment success rate (%): 72

<APC-A>: hCD33<PE-Cy7-A>: hGPR56

<PE-A>: hCD34

0 102 103 104 105

0102

103

104

105

<FITC-A>: hCD45

0102

103

104

105

hCD

34

0 102 103 104 105

hCD

45

mCd45.1 hCD38

hGPR

56

hCD45

0102

103

104

105

0 102 103 104 105

0102

103

104

105

0 102 103 104 105

hCD

33

hCD45

AML marker expression(Bone marrow and spleen)


Preclinical testing of imetelstat in AML PDX


NSGS

Primary or serial AML patient sample

(CD3 depletion)

(Check peripheral blood donor chimerism)Randomize

Imetelstat (15 mg / kg bw) or PBS tiw ip

EngraftmentAML symptoms

0

20

40

60

80

100

Age

at d

iagn

osis

[y

ears

]

Age at diagnosis

0

5

10

15

# A

ML

patie

nt s

ampl

es

ELN prognosis(Doehner et al., Blood 2010

Alpermann et al., Blood 2011)

favorableintermediate 1intermediate 2adverse

0

5

10

15

# A

ML

patie

nt s

ampl

es

Gender

femalemale


Imetelstat prolongs overall survival in AML PDX

0 100 200 300

0

50

100

Overall survival

Days post-start of treatment

Pe

rc

en

t s

urv

iva

l

PBS

Imetelstat

Median survival:

PBS: 83

Imetelstat: 153

p < 0.0001


Imetelstat suppresses AML expansion in 14 out of 15 PDX

0 50 100 1500

10

20

30

40

50

0 5 10 15 20 250

20

40

60

0 50 1001502000

20

40

60

80

0 20 40 60 800

10

20

30

40

0 50 100 1500

5

10

15

20

25

0 20 40 600

2

4

6

8

10

0 50 100 1500

20

40

60

0 50 100 1500

20

40

60

0 20 40 600

20

40

60

80

0 50 1000

20

40

60

80

0 10 20 30 400

20

40

60

80

0 50 100 1500

20

40

60

80

0 10 20 30 40 500

10

20

30

40

0 10 20 30 400

2

4

6

8

0 10 20 30 40 500

20

40

60

80

100

Perip

hera

l blo

od d

onor

chi

mer

ism

[%]


PBSImetelstat

AML expansion

0 100 200 300

0

50

100

Overall survival


Pe

rc

en

t s

urv

iva

l

PBS

Imetelstat

Median survival:

PBS: 83

Imetelstat: 153

p < 0.0001


AML PDX can be separated into two groups with distinct response to imetelstat therapy

0 100 200 3000

50

100

Sustained responders


Perc

ent s

urvi

val p <0.0001

0 100 200 3000

50

100


Perc

ent s

urvi

val

Poor responders

PBSImetelstatp = 0.3351

0

2

4

6

8

10

# A

ML

patie

nt s

ampl

es

ELN prognosis(Sustained responders)

favorable

intermediate 1intermediate 2adverse

0

2

4

6

8

10

# A

ML

patie

nt s

ampl

es

ELN prognosis(Poor responders)


Next generation sequencing reveals baseline mutations in AML patient samples

HemePACT assay in collaboration with Stanley Chun-Wei Lee and Omar Abdel-Wahab, MSKCC

NPM1

DNMT3AID

H2FLT3

NRASKRAS

TET2ETV6

EZH2

FANCMMKI67

PTPN11

RAD21

SRSF2WT1

APC

ARID1B

ASXL1AXL

BCOR

BCORL1

BRCA1BRD4

CD36

CXCR4

ECT2LESR1

ETS1

FANCFJA

K3

KDM2B

MAGED1MSH2

MSH3

PARK2

PLCG2

PTPRD

RAD54L

RUNX1

SMC1ASMO

STAG1TLL2

TRAF2

U2AF1

ZNF703

0

2

4

6

8

# A

ML

patie

nt s

ampl

es


The identity and distribution of mutations in selected PDX reflects larger AML cohorts

Papaemmanuil et al., NEJM 2016

NPM1

DNMT3AID

H2FLT3

NRASKRAS

TET2ETV6

EZH2

FANCMMKI67

PTPN11

RAD21

SRSF2WT1

APC

ARID1B

ASXL1AXL

BCOR

BCORL1

BRCA1BRD4

CD36

CXCR4

ECT2LESR1

ETS1

FANCFJA

K3

KDM2B

MAGED1MSH2

MSH3

PARK2

PLCG2

PTPRD

RAD54L

RUNX1

SMC1ASMO

STAG1TLL2

TRAF2

U2AF1

ZNF703

0

2

4

6

8

# A

ML

patie

nt s

ampl

es

n engl j med 374;23 nejm.org June 9, 2016 2211

Genomic Classification and Prognosis in Leukemia

number alterations (18), clinical variables (11), demographic characteristics (2), treatment (3), nuisance (4; nuisance variables are other or miss-ing variables, such as the trial a patient was en-rolled in, the year a patient entered the clinical

trial, and whether cytogenetic data were missing), and gene–gene interaction terms, defined as nonadditive effects on survival between two genes when both are mutated (126). We used an expectation-maximization algorithm to estimate

Figure 1. Landscape of Driver Mutations in Acute Myeloid Leukemia (AML).

Panel A shows driver events in 1540 patients with AML. Each bar represents a distinct driver lesion; the lesions include gene mutations, chromosomal aneuploidies, fusion genes, and complex karyotypes. The colors in each bar indicate the molecular risk according to the European LeukemiaNet (ELN) classification. Panel B shows the distribution of samples and overlap (cross-sections) across molecular subgroups (vertical bars). Patients who had no driver mutations and those who had driver mutations but did not meet the criteria for any specific class are also included. The number at the top of each column is the number of patients assigned solely to the designated class; the numbers of patients meeting criteria for two or more classes are shown at the intersection of classes.

B

A

No.

of U

niqu

e Pa

tient

s w

ith D

river

Mut

atio

n

500

300

100

400

200

0

20

15

10

5

0

FLT3

NPM1

DNMT3A

NRAS

comple

xTET2

!7/7q+8/

8qID

H2

CEBPA

RUNX1

PTPN11!5/

5qID

H1TP53

SRSF2

inv(16)

MLL

WT1KRAS

!17/1

7p

ASXL1 KIT

STAG2

t(15;1

7)

t(8;21

)

t(11q

23;x)

RAD21 !9qab

n3qEZH2

PHF6 !YSF3

B1+21 CBL

U2AF1BCOR

GATA2NF1

!20/2

0q

!18/1

8q

inv(3)/t

(3;3)

EP300ETV6

+13!4/

4qM

YC+22

!12/1

2p

No. of Patients

+11/1

1qt(6

;9)

KDM5AM

LL2

ZRSR2JA

K2

CREBBP

KDM6AM

LL3BRAF

FBXW

7ATRX

CUX1RB1

MPL

PRPF40BPTEN

CDKN2AGNAS

MLL

5SF1

U2AF2CBLB

IKZF1

SF3A1

SH2B3

166

81 1 1

60

60 2 1

44 1 11

20 1 2

15

418 2 8 6

66 3

199 16

18

275

166

62

2 3inv(16) t(1

5;17) t(8

;21)

inv(3) t(6

;9)NPM

1 CEBPAbia

llelic

TP53–a

neuploidy

Chrom

atin–s

pliceo

som

e

IDH2R

172

No clas

s No drivers

detecte

d

MLL

fusio

n

inv(16)

t(15;17)

t(8;21)

inv(3)

t(6;9)

NPM1CEBPAbiallelic

TP53–aneuploidy

Chromatin–spliceosome

IDH2R172

No class

No drivers detected

MLL fusion

ELN intermediate-I riskELN favorable risk ELN intermediate-II risk

ELN adverse risk

ELN risk not available

1 5 10 15 >15

The New England Journal of Medicine Downloaded from nejm.org on December 4, 2016. For personal use only. No other uses without permission.

Copyright © 2016 Massachusetts Medical Society. All rights reserved.

HemePACT assay in collaboration with Stanley Chun-Wei Lee and Omar Abdel-Wahab, MSKCC


Imetelstat response is correlated with a distinct mutational landscape

NP

M1

DN

MT

3A

IDH

2

KR

AS

EZ

H2

PT

PN

11

SR

SF

2

Su

sta

ined

resp

on

ders

Po

or

resp

on

ders

Mutations occuring in

both sustained and

poor responders



NP

M1

DN

MT

3A

IDH

2

KR

AS

EZ

H2

PT

PN

11

SR

SF

2

FLT

3

NR

AS

TE

T2

RA

D21

AR

ID1B

CD

36

EC

T2L

JA

K3

KD

M2B

MA

GE

D1

MS

H2

MS

H3

PA

RK

2

PT

PR

D

RA

D54L

RU

NX

1

SM

C1A

STA

G1

ZN

F703

Su

sta

ined

resp

on

ders

Po

or

resp

on

ders

Mutations occuring in

both sustained and

poor responders

Mutations occuring

exclusively

in sustained responders



NPM

1DN

MT3

AID

H2KR

ASEZ

H2PT

PN11

SRSF

2FL

T3NR

ASTE

T2RA

D21

ARID

1BCD

36EC

T2L

JAK3

KDM

2BM

AGED

1M

SH2

MSH

3PA

RK2

PTPR

DRA

D54L

RUNX

1SM

C1A

STAG

1ZN

F703

ETV6

FANC

MM

KI67

WT1

APC

ASXL

1AX

LBC

OR

BCO

RL1

BRCA

1BR

D4CX

CR4

ESR1

ETS1

FANC

FPL

CG2

SMO

TLL2

TRAF

2U2

AF1

Sust

aine

d re

spon

ders

Poor

resp

onde

rs

Mutations occuring in both sustained and

poor responders

Mutations occuring exclusively

in sustained responders

Mutations occuring exclusively

in poor responders


Gene ontology analysis of mutually exclusive mutations reveals distinct molecular pathways

0 50 100 150 200 250

cell cycledeath

cell deathprogrammed cell death

apoptosiscellular response to stress

cell cycle processDNA metabolic process

response to abiotic stimulusresponse to DNA damage stimulus

DNA repairresponse to radiation

chromosome segregationDNA recombination

response to ionizing radiationdouble-strand break repair

postreplication repair

Fold enrichment


0

regulation of cell deathregulation of programmed cell death

chromosome organizationpositive regulation of developmental process

immune system developmentPathways in cancer

pattern specification processpositive regulation of cell differentiationregulation of myeloid cell differentiation

10 20 30Fold enrichment

Poor responders



0 50 100 150 200 250

cell cycledeath









Fold enrichment


0






Poor responders

1.  DNA repair



0 50 100 150 200 250

cell cycledeath









Fold enrichment


0






Poor responders

1.  DNA repair

2.  Cell cycle



0 50 100 150 200 250

cell cycledeath









Fold enrichment


0






Poor responders

1.  DNA repair

2.  Cell cycle

3.  Development and differentiation



0 50 100 150 200 250

cell cycledeath









Fold enrichment


0






Poor responders

1.  DNA repair

2.  Cell cycle

3.  Development and differentiation

4.  Pathways in cancer

Imetelstat induces DNA damage and loss of quiescence in LSC in vivo



NSGS

AML patient sample

(Check peripheral blood donor chimerism)Randomize


Endpoint analysis at disease onset of PBS group

G0

PBS

Imete

lsta

t

0

5

10

15

20

25

LS

C in

G0

[%

]

p = 0.0002

AML-16

AML-5

AML-18

0 102

103

104

105

aH2AX

PBS

Imetelstat

aH2AX

aH2

AX

MF

I in

G1

LS

C

[no

rm

alize

d to

PB

S]

PBS

Imete

lsta

t

0.0

0.5

1.0

1.5

p < 0.0001


Modelling normal human hematopoiesis

Sublethal

irradiation

(2.8 Gy)

NSG

Cord blood donor sample

CD34

enrichment


Human hematopoiesis phenotype

endpoint analysis

10 weeks


Imetelstat primarily depletes B lymphocytes

Sublethal

irradiation

(2.8 Gy)

NSG


CD34

enrichment



endpoint analysis

10 weeks

Donor chimerism(Spleen)

Donor 1: P

BS

Donor 1: Im

etelst

at

Donor 2: P

BS

Donor 2: Im

etelst

at0

20

40

60

80

100

Dono

r chi

mer

ism

[%]

** ****

B cells(Spleen)

Donor 1: P

BS

Donor 1: Im

etelst

at

Donor 2: P

BS

Donor 2: Im

etelst

at0

20

40

60

80

100

CD

19+

[% o

f via

ble

CD

45+] *

p = 0.08

Myeloid cells (Spleen)

Donor 1: P

BS

Donor 1: Im

etelst

at

Donor 2: P

BS

Donor 2: Im

etelst

at0

5

10

15

CD

33+

[% o

f via

ble

CD

45+] * p = 0.0667


Human cord blood - derived stem cells are preserved during imetelstat treatment

Sublethal

irradiation

(2.8 Gy)

NSG


CD34

enrichment



endpoint analysis

10 weeks

Donor chimerism(Bone marrow)

Donor 1: P

BS

Donor 1: Im

etelst

at

Donor 2: P

BS

Donor 2: Im

etelst

at0

20

40

60

80

100

Dono

r chi

mer

ism

[%]

** ****

HSC

(Bone marrow)

Donor 1: PBS

Donor 1: Im

ete

lsta

t

Donor 2: PBS

Donor 2: Im

ete

lsta

t

0

1

2

3

CD

34

+C

D3

8- fr

om

CD

45

+ %

H2AX in HSC

(Bone marrow)

Donor 1: PBS

Donor 1: Im

ete

lsta

t

Donor 2: PBS

Donor 2: Im

ete

lsta

t

0

500

1000

1500

MF

I

H2

AX

in

G1

CD

34

+C

D3

8-


Summary: Preclinical efficacy of imetelstat in AML PDX

•  Imetelstat is effective in a subgroup (60%) of AML patient samples

•  Imetelstat prevents expansion and prolongs overall survival in AML PDX (PBS: 83 days; Imetelstat: 153 days post-start of treatment)

•  Sustained responses to imetelstat are correlated with favorable cytogenetics, mutational profiles of DNA damage and activation of DNA damage response pathways

•  This study has generated preclinical data to inform clinical trials and provide a precision approach to targeted therapies in patients with AML

QIMR Berghofer Medical Research Institute Gordon and Jessie Gilmour Leukaemia Research Lab -  Steven Lane -  Brad Wackrow -  Axia Song -  Amy Porter -  Joanne Sutton -  Sebastien Jacquelin -  Therese Vu -  Rebecca Austin -  Emma Dishington -  Solene Guignes -  Jasmin Straube -  FACS Core: Grace, Paula, Michael -  Animal House: Sue, Jonathan, Dave -  Statistics: Leesa, Louise, Mandy, Peter

Royal Brisbane and Women’s Hospital -  Patients and health professionals

Acknowledgements


Memorial Sloan Kettering Institute -  Omar Abdel-Wahab -  Stanley Chun-Wei Lee The University of Queensland Diamantina Institute, Translational Research Institute -  Andrew Moore

The University of New South Wales -  Richard Lock The University of South Australia -  Hamish Scott

Funding NHMRC Leukaemia Foundation of Australia Cure Cancer Australia Rio Tinto Ride to Conquer Cancer Janssen (research funding agreement)

The preclinical efficacy of a novel telomerase inhibitor, imetelstat ... · Baerlocher et al., NEJM...

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