targeting rb loss in osteosarcoma - bcrt.org.uk fileretinoblastoma protein biology..predicts...

Targeting RB1 loss in osteosarcoma

Sibylle Mittnacht

UCL Cancer Institute

London

Retinoblastoma tumour suppressor loss in Osteosarcoma

FREQUENT: High percentage of OS (20-70 %) have suspected deleterious mutations affecting RB1

EARLY:RB mutations present at diagnosisIncreased OS risk in individual with germ-line deleterious RB1 mutationRB1 mutations truncal event

BAD:RB1 LOH or genomic alteration predicts poor prognosis

Retinoblastoma protein biology

..predicts phenotypic liabilities that could provide synthetic sick/ synthetic lethal opportunities?

The consequences of RB1 inactivation

(Dyson,2016)

Increased stem-ness

Changes in signalling pathways

Reduced chromosome cohesion

Mitochondrial defects

Deregulated transcription

Defects in cell cycle exit

Defects in cell cycle response to DNA damage

Impaired senescence

Altered nucleotide pool

Altered replication dynamicChanges in Redox control

Altered sensitivity to apoptoticstimuli

DS DNA repair defect

A “basket cell line panel” identifies selective pharmacological sensitivities associated with RB1

defect

ATM inhibitor (KU60019)

0.01 0.1 1 10 1000

50

100

150

log[KU60019] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)

0.01 0.1 1 10 1000

50

100

150

log[Olaparib] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)

PARP inhibitor (Olaparib)

0.01 0.1 1 10 1000

50

100

150

log[NU7441] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-) H1299 (RB+)

H1975 (RB+)

H2122 (RB+)

SAOS2 (RB-) U2OS (RB+)

DNAPK inhibitor (NU7441)

ATR inhibitor (AZD6738)

0.01 0.1 1 10 1000

50

100

150

log[AZD6738] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)

0.01 0.1 1 10 1000

50

100

150

log[VE821] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

U2OS (RB+)

A

D

B C

G

p value: 0.231 p value: 0.011 p value: 0.521

p value: 0.0103

p value: 0.0172

RB- RB+200

300

400

Area U

nd

er C

urve

AZD6738

P value 0.0103

*

E

HATR inhibitor

(VE821)

RB- RB+200

300

400

500

Area U

nd

er C

urve

VE821

*

Figure 1

F

Chk1 (pS345)

- + + + + + IR

GAPDH

Chk2 (pT68)

DNA-PKcs (pS2056)

ATM

DNA-PKcs

Chk2

Chk1

ATM (pS1981)

H1975

d5

via

ble

cel

ls (

Res

azu

rin

), r

elat

ive

Log conc. (mM)

P value (AUC): 0.231


0.01 0.1 1 10 1000

50

100

150

log[KU60019] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)

0.01 0.1 1 10 1000

50

100

150

log[Olaparib] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)


0.01 0.1 1 10 1000

50

100

150

log[NU7441] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-) H1299 (RB+)

H1975 (RB+)

H2122 (RB+)




0.01 0.1 1 10 1000

50

100

150

log[AZD6738] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)

0.01 0.1 1 10 1000

50

100

150

log[VE821] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

U2OS (RB+)

A

D

B C

G


p value: 0.0103

p value: 0.0172

RB- RB+200

300

400

Area U

nd

er C

urve

AZD6738

P value 0.0103

*

E

HATR inhibitor

(VE821)

RB- RB+200

300

400

500

Area U

nd

er C

urve

VE821

*

Figure 1

F

Chk1 (pS345)

- + + + + + IR

GAPDH

Chk2 (pT68)

DNA-PKcs (pS2056)

ATM

DNA-PKcs

Chk2

Chk1

ATM (pS1981)

H1975


0.01 0.1 1 10 1000

50

100

150

log[KU60019] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)

0.01 0.1 1 10 1000

50

100

150

log[Olaparib] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)


0.01 0.1 1 10 1000

50

100

150

log[NU7441] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-) H1299 (RB+)

H1975 (RB+)

H2122 (RB+)




0.01 0.1 1 10 1000

50

100

150

log[AZD6738] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)

0.01 0.1 1 10 1000

50

100

150

log[VE821] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

U2OS (RB+)

A

D

B C

G


p value: 0.0103

p value: 0.0172

RB- RB+200

300

400

Area U

nd

er C

urve

AZD6738

P value 0.0103

*

E

HATR inhibitor

(VE821)

RB- RB+200

300

400

500

Area U

nd

er C

urve

VE821

*

Figure 1

F

Chk1 (pS345)

- + + + + + IR

GAPDH

Chk2 (pT68)

DNA-PKcs (pS2056)

ATM

DNA-PKcs

Chk2

Chk1

ATM (pS1981)

H1975 ATM inhibitor (KU60019)

0.01 0.1 1 10 1000

50

100

150

log[KU60019] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)

0.01 0.1 1 10 1000

50

100

150

log[Olaparib] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)


0.01 0.1 1 10 1000

50

100

150

log[NU7441] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-) H1299 (RB+)

H1975 (RB+)

H2122 (RB+)




0.01 0.1 1 10 1000

50

100

150

log[AZD6738] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

H1299 (RB+)

U2OS (RB+)

0.01 0.1 1 10 1000

50

100

150

log[VE821] (µM)

Cell P

rolife

ration (

% C

ontr

ol)

H2009 (RB-)

H82 (RB-)

H446 (RB-)

SAOS2 (RB-)

H1975 (RB+)

H2122 (RB+)

U2OS (RB+)

A

D

B C

G


p value: 0.0103

p value: 0.0172

RB- RB+200

300

400

Area

Un

der C

urve

AZD6738

P value 0.0103

*

E

HATR inhibitor

(VE821)

RB- RB+200

300

400

500

Area U

nd

er C

urve

VE821

*

Figure 1

F

Chk1 (pS345)

- + + + + + IR

GAPDH

Chk2 (pT68)

DNA-PKcs (pS2056)

ATM

DNA-PKcs

Chk2

Chk1

ATM (pS1981)

H1975

RB 1 defective (H2009, H82, SF539, H446, SAOS2)

P value (AUC):0.011

P value (AUC):0.010

P value (AUC):0.521

Log conc. (mM) Log conc. (mM) Log conc. (mM)

RB 1 normal (H1975, H2122, SF259, H1299, U2OS)

PARP inhibitor(Olapaprib)

DNAPK inhibitor(Nu7441)

ATR inhibitorAZD6738

ATM inhibitor KU60019

13-123-Oval-Clothes-Basket-e1441275948714.jpg (JPEG Image... https://www.kosmopolitan.co.uk/wp-content/uploads/2015/08/13...

1 of 1 30/01/2019, 17:52

RB1 defect predicts hypersensitivity to PARP inhibitors in OS

0.01 0.1 1 10 1000

50

100

Concentration (uM)

Percen

tag

e o

f V

iab

le C

ells

Talazoparib all cell lines SOAS (RC)

U2OS (RC)

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)

G292

KPD

CAL72

LM7

SJSAOSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 0.1 1 10 1000

50

100

Concentration uM

SOAS (RC)

U2OS (RC)

CAL72

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)

Niraparib all cell lines

G292

Percentage of Viable cells

LM7

KPD

SJSAOSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 0.1 1 10 1000

50

100

Concentration (uM)

Pe

rc

en

tag

e o

f V

iab

le C

ells

Veliparib all cell lines

SOAS (RC)

U2OS (RC)

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)

G292

KPD

CAL72

LM7

SJSA/OSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 1 1000

50

100

Concentration (uM)

Perce

ntag

e o

f V

iab

le C

ells

Olaparib all cell lines N=2 HU09

NY

OHSN

SAOS (sanger)

143b

MHM

CAL72

HU03N1

MG63

OS25HAL

HOS

SJSA/OSA

HOSMNNG

U2OS (sanger)

U2OS (RC)

SAOS (RC)

G292

KPD

LM7

CAPAN1

IC50

HU09

18.43

NY

47.62

OHSN

8.135

SAOS (sanger)

17.11

143b

49.9

MHM

75.64

CAL72

99.06

HU03N1

21.91

MG63

19.77

OS25HAL

81.83

HOS

35.26

SJSA/OSA

78.08

HOSMNNG

52.7

U2OS (sanger)

201.9

U2OS (RC)

~ 114.6

SAOS (RC)

5.322

G292

77.35

KPD

75.2

LM7

15.04

CAPAN1

12.44

RB-

RB+

0

100

200

300

400

500

Osteosarcoma

AU

C

RB-

RB+

P value 0.0480

*

Talazoparib AUC

RB-

RB+

200

250

300

350

400

Osteosarcoma

AU

C

RB-

RB+

P value 0.0472

*

Niraparib AUC

RB-

RB+

350

400

450

500

Osteosarcoma

AU

C

RB-

RB+

P value 0.0024

**

Veliparib AUC

RB-

RB+

250

300

350

400

450

500

Osteosarcoma

AU

C

Olaparib AUC Two repeats

RB-

RB+

P value <0.0001

****

Conclusion:

0.01 0.1 1 10 1000

50

100

Concentration (uM)

Percen

tag

e o

f V

iab

le C

ells


U2OS (RC)

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)

G292

KPD

CAL72

LM7

SJSAOSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 0.1 1 10 1000

50

100

Concentration uM

SOAS (RC)

U2OS (RC)

CAL72

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)


G292


LM7

KPD

SJSAOSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 0.1 1 10 1000

50

100

Concentration (uM)

Percen

tag

e o

f V

iab

le C

ells


SOAS (RC)

U2OS (RC)

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)

G292

KPD

CAL72

LM7

SJSA/OSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 1 1000

50

100

Concentration (uM)

Perc

en

ta

ge o

f V

iab

le C

ells


NY

OHSN

SAOS (sanger)

143b

MHM

CAL72

HU03N1

MG63

OS25HAL

HOS

SJSA/OSA

HOSMNNG

U2OS (sanger)

U2OS (RC)

SAOS (RC)

G292

KPD

LM7

CAPAN1

IC50

HU09

18.43

NY

47.62

OHSN

8.135

SAOS (sanger)

17.11

143b

49.9

MHM

75.64

CAL72

99.06

HU03N1

21.91

MG63

19.77

OS25HAL

81.83

HOS

35.26

SJSA/OSA

78.08

HOSMNNG

52.7

U2OS (sanger)

201.9

U2OS (RC)

~ 114.6

SAOS (RC)

5.322

G292

77.35

KPD

75.2

LM7

15.04

CAPAN1

12.44

RB-

RB+

0

100

200

300

400

500

Osteosarcoma

AU

C

RB-

RB+

P value 0.0480

*

Talazoparib AUC

RB-

RB+

200

250

300

350

400

Osteosarcoma

AU

C

RB-

RB+

P value 0.0472

*

Niraparib AUC

RB-

RB+

350

400

450

500

OsteosarcomaA

UC

RB-

RB+

P value 0.0024

**

Veliparib AUC

RB-

RB+

250

300

350

400

450

500

Osteosarcoma

AU

COlaparib AUC Two repeats

RB-

RB+

P value <0.0001

****

Conclusion:

0.01 0.1 1 10 1000

50

100

Concentration (uM)

Percen

tag

e o

f V

iab

le C

ells


U2OS (RC)

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)

G292

KPD

CAL72

LM7

SJSAOSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 0.1 1 10 1000

50

100

Concentration uM

SOAS (RC)

U2OS (RC)

CAL72

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)


G292


LM7

KPD

SJSAOSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 0.1 1 10 1000

50

100

Concentration (uM)

Percen

tag

e o

f V

iab

le C

ells


SOAS (RC)

U2OS (RC)

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)

G292

KPD

CAL72

LM7

SJSA/OSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 1 1000

50

100

Concentration (uM)

Percen

tag

e o

f V

iab

le C

ells


NY

OHSN

SAOS (sanger)

143b

MHM

CAL72

HU03N1

MG63

OS25HAL

HOS

SJSA/OSA

HOSMNNG

U2OS (sanger)

U2OS (RC)

SAOS (RC)

G292

KPD

LM7

CAPAN1

IC50

HU09

18.43

NY

47.62

OHSN

8.135

SAOS (sanger)

17.11

143b

49.9

MHM

75.64

CAL72

99.06

HU03N1

21.91

MG63

19.77

OS25HAL

81.83

HOS

35.26

SJSA/OSA

78.08

HOSMNNG

52.7

U2OS (sanger)

201.9

U2OS (RC)

~ 114.6

SAOS (RC)

5.322

G292

77.35

KPD

75.2

LM7

15.04

CAPAN1

12.44

RB-

RB+

0

100

200

300

400

500

Osteosarcoma

AU

C

RB-

RB+

P value 0.0480

*

Talazoparib AUC

RB-

RB+

200

250

300

350

400

Osteosarcoma

AU

C

RB-

RB+

P value 0.0472

*

Niraparib AUC

RB-

RB+

350

400

450

500

Osteosarcoma

AU

C

RB-

RB+

P value 0.0024

**

Veliparib AUC

RB-

RB+

250

300

350

400

450

500

Osteosarcoma

AU

C


RB-

RB+

P value <0.0001

****

Conclusion:

0.01 0.1 1 10 1000

50

100

Concentration (uM)

Percen

tag

e o

f V

iab

le C

ells


U2OS (RC)

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)

G292

KPD

CAL72

LM7

SJSAOSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 0.1 1 10 1000

50

100

Concentration uM

SOAS (RC)

U2OS (RC)

CAL72

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)


G292


LM7

KPD

SJSAOSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 0.1 1 10 1000

50

100

Concentration (uM)

Percen

ta

ge o

f V

iab

le C

ells


SOAS (RC)

U2OS (RC)

SAOS (sanger)

HU03N1

NY

HOS

HOSMNNG

OHSN

U2OS (sanger)

G292

KPD

CAL72

LM7

SJSA/OSA

MG63

143b

HU09

MHM

OS25HAL

CAPAN1

0.01 1 1000

50

100

Concentration (uM)

Percen

tag

e o

f V

iab

le C

ells


NY

OHSN

SAOS (sanger)

143b

MHM

CAL72

HU03N1

MG63

OS25HAL

HOS

SJSA/OSA

HOSMNNG

U2OS (sanger)

U2OS (RC)

SAOS (RC)

G292

KPD

LM7

CAPAN1

IC50

HU09

18.43

NY

47.62

OHSN

8.135

SAOS (sanger)

17.11

143b

49.9

MHM

75.64

CAL72

99.06

HU03N1

21.91

MG63

19.77

OS25HAL

81.83

HOS

35.26

SJSA/OSA

78.08

HOSMNNG

52.7

U2OS (sanger)

201.9

U2OS (RC)

~ 114.6

SAOS (RC)

5.322

G292

77.35

KPD

75.2

LM7

15.04

CAPAN1

12.44

RB-

RB+

0

100

200

300

400

500

Osteosarcoma

AU

C

RB-

RB+

P value 0.0480

*

Talazoparib AUC

RB-

RB+

200

250

300

350

400

Osteosarcoma

AU

C

RB-

RB+

P value 0.0472

*

Niraparib AUC

RB-

RB+

350

400

450

500

Osteosarcoma

AU

C

RB-

RB+

P value 0.0024

**

Veliparib AUC

RB-

RB+

250

300

350

400

450

500

Osteosarcoma

AU

C


RB-

RB+

P value <0.0001

****

Conclusion:

RB1 defective (SAOS2, OSHN, HUO3N1, NY, LM7, HUO9)

RB1 normal (U2OS, CAl72, HOS, HOSMMG, KPD, G292, SJSA, MG63, 143B, MHM, OS25HAL)


1 of 1 30/01/2019, 17:52

Talazoparib OlaparibNiraparib Veliparib

d5

via

ble

cel

ls (

Res

arzu

rin

),

rela

tive

RB1 normal, BRCA2 defective (CAPAN1)

Log (conc.) mM

AU

C

AU

C

AU

C

AU

C

RB-

RB+

250

300

350

400

450

AU

C


RB-

RB+

P value <0.0001

****

Carlosfigurewherehehasonlyrepeat2andrepeat3 Thenewcombinedfigurewithall3repeats(includingrepeat1whichseemsthatitcompromisesthesignificanceofthedata)-s?llsignificantbutonestar

RB- RB+

0

100

200

300

400

500

Olaparib n=3 repeats

Area U

nd

er C

urve RB-

RB+

p value = 0.0231

*

RB- RB+

0

100

200

300

400

500

Talazoparib n=2

Area U

nd

er C

urve RB-

RB+

p value = 0.0054

**

Thenewcombinedfigurewithall2repeats

RB- RB+

360

380

400

420

440

460

Veliparib combined n=2

Area U

nd

er C

urve RB-

RB+

p value < 0.0001

****


RB- RB+

0

100

200

300

400

500

Talazoparib n=2

Area U

nd

er C

urve RB-

RB+

p value = 0.0054

**


RB- RB+

360

380

400

420

440

460

Veliparib combined n=2

Area U

nd

er C

urve RB-

RB+

p value < 0.0001

****

Thenewcombinedfigurewithall2repeatsThenewcombinedfigurewithall2repeats

RB- RB+

200

250

300

350

400

Niraparib combined n=2

Area U

nd

er C

urve RB-

RB+

p value = 0.0033

**

Log (conc.) mM Log (conc.) mM Log (conc.) mM

Olaparib sensitivity in RB defective lines well below the clinically achievable concentration

mM

HU09 0.565 ± 0.03

SAOS (RC) 0.522 ± 0.53

NY 0.134 ± 0.01

LM7 0.110 ± 0.01

OHSN 0.051 ± 0.03

HU03N1 0.043 ± 0.02

mM

U2OS § §

143b § §

CAL72 § §

MHM § §

HOSMNNG 3.026 ±0.31

HOS 1.827 §

CAPAN1 1.414 ±0.29

SJSA/OSA) 1.334 ±0.42

OS25HAL 1.311 ±0.010

KPD 0.405 ±0.024

G292 0.229 ±0.12

_RB1 defective (n=2)

_RB1 normal (n=2)

Clo

no

gen

icac

tivi

ty (

rela

tive

)

Log 10 Olaparib (mM)RB defective RB normal

0

100

200

300

400

expt 1 and 2

clo

no

gen

ic a

cti

vit

y (

AU

C)

RB defective

RB normal

IC 50 (±STDV) n=2

****

RB

1 d

efec

tive

RB

1 n

orm

al

AU

C

0.1 1 20

50

100

log [Olaparib] (uM)

Co

lon

y G

row

th (

% C

on

tro

l)

Olaparib effect on Clonogenic Survival

KPDOHSN

HU03N1 G292

SAOS2

NY

U2OS

143b

HOSMNNG

CAL72

HOS

MHM

MG63

SJSA/OSA

CAPAN1

(BRCA- Mut

LM7

HU09

KPD

G292

U2OS

143b

HOSMNNG

CAL72

HOS

MHM

MG63

SJSA/OSA

CAPAN1

(BRCA- Mut)

RB-

RB+

0

50

100

150

200

Osteosarcoma

Are

a U

nd

er

Cu

rve

Clonogenic Assays

RB-

RB+

***

Conclusion:

RB1 deletion in RB normal OS causes Olaparibhypersensitivty

•

Em

pty

gip

z

sh

RB

1

shR

B2

sh

RB

4

sh

RB

3

shR

B5

sh

RB

6

•

Em

pty

gip

z

sh

RB

1

sh

RB

2

sh

RB

4

sh

RB

3

sh

RB

5

sh

RB

6

Vec

tor

Vec

tor

LentiCRISPR v2 GIPZ

CR

ISP

R 1

CR

ISP

R 1

CR

ISP

R 1

RB1

GAPDH

shR

B1

-1

shR

B1

-2

shR

B1

-1

shR

B1

-3

shR

B1

-4

shR

B1

-5

shR

B1

-6

CAL72 (RB1 normal)

10-3 10-2 10-1 100 20

50

100

log [Olaparib] (uM)C

olo

ny G

row

th (

% C

on

tro

l)

shCAL72

unmodifiedshRB1

shRB2Vector

shRB5

shRB6

shRB1 (1,2,5,6)

Unmodified, vector


1 of 1 30/01/2019, 17:52

RB1 defective (Cal72 shRB1-1, Cal72 shRB1-2, Cal72 shRB1-5, Cal72 shRB1-6)

RB1 normal (Cal72 unmodified, CAL72 GIPZ Vector)

Clo

no

gen

icac

tivi

ty (

rela

tive

)

Log (Olaparib) mM

IC 50

mM

0.81 shRB1-6

0.56 shRB1-2

0.36 shRB1-5

0.34 shRB1-1

§ unmodified

§ GIPZ Vector

Translating bench to bedside

Conceptual studies Mechanism PARP inhibitor hypersensitivityRoutes to resistance/ nonresponseOther Opportunities?

Preclinical proof of conceptPatient near, patient relevant

How to ID patients with RB defective cancers in the clinic?

Where/ how to move this into clinical practiceChallenges

Shape and function of RB1 1998- 2003: The Pocket taking Shape

Cyclin fold

A

Cyclin fold

B LXCXE

LXCXE

} {

} {

E2Fct

E2Fct

2007: pRB N domain reveals its form

Cyclin fold

A

Cyclin fold

B

} {

} {

(in press) Bear

Bear

No E2Fct !!!

Mutations in RB

Mittnacht lab.(UCL-CI)

Georgia Zoumpoulidou

Caterina Mancusi

Carlos Mendosa

Ritika Ahmed

Sandra Strauss (Co-lead, UCL CI and

UCLH)

Collaborators

Chris Lord (ICR London)

15.jpg (JPEG Image, 880 × 320 pixels) https://www.simpsonyork.co.uk/wp-content/uploads/2013/02/15.jpg

1 of 1 30/01/2019, 15:14

Acknowledgement

International Osteosarcoma Research Symposium

In Memory of Frankie Biggs

31st January 2019

Goodenough College, Mecklenburgh Square, London WC1N 2AB

Retinoblastoma protein biology

..predicts phenotypic liabilities of RB loss that could provide synthetic sick / synthetic lethal opportunities?

RB1

DNA repair

Stemness

Mitotic Fidelity

Apoptosis

G1/ S Cell cycle transit

Replicationfidelity

targeting rb loss in osteosarcoma - bcrt.org.uk fileretinoblastoma protein biology..predicts...

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