Sarcoma Research Laboratory University of Texas, MD Anderson Cancer Center

PARP Inhibitors for the treatment of MPNST

Christine Kivlin, Roman Belousov, Gonzalo Lopez, Quan-Sheng Zhu, Kai-Lieh Huang, Davis Ingram, Keila E. Torres, Alexander J. Lazar, Raphael E. Pollock, Dina Lev

Malignant Peripheral Nerve Sheath Tumor (MPNST)

• Accounts for 3–10% of soft tissue sarcomas • Up to 1,200 new cases in the U.S. per year • High metastatic potential • Neurofibromatosis type I (NF1)-related cases (50%) and sporadic (50%) • Associated with a precursor lesion (NF1-related, deep neurofibromata)

MPNST treatment and outcome

• Surgical excision is the mainstay of treatment • Radiation/chemotherapy =?

• High rate of local and systemic recurrence Zou

et al.,

2009

There is a critical need for novel effective therapies in MPNST

PARP inhibitors for the treatment of cancer

• PARPi have been used in clinical trials over the past few years for common cancers, role for sarcoma unknown • Tumors with DNA repair defects are most highly sensitive

The goal of our studies was to determine the effects of PARP inhibitors on MPNST in vitro and in vivo

Experimental Models

Time (weeks)

Lopez

et al.,

2011

PARPi treatment reduces PARP activity

1,4 1,6 0,8 1 0,8 0,6 0,6 0 0

AZD2281

Olaparib (AstraZeneca)

ABT888

Veliparib (Abbott Laboratories)

BSI201

Iniparib (Sanofi Aventis)

Decrease in cell proliferation with AZD2281

Control Cell lines 96 hour treatment MDA-231 724 MDA-436 AZD2281 (uM)

Decrease in cell proliferation with AZD2281

MPNST Cell Lines 96 hour treatment NSC 724 26T ST88 462 AZD2281 (uM)

1,2 1 0,8 0,6 0,4 0,2 0

AZD2281 treatment decreases colony forming ability

MPNST 724 control 0.625

1.25

2.5

5.0

10.0

control 0.6125

1.25

2.5

AZD2281 (uM) 5.0 10.0

AZD2281 causes G2/M cell cycle arrest

control G1 S G2 MPNST 724 24 hour treatment 2.5uM AZD G2 G1 S 5.0uM AZD G2 G1 S 10.0uM AZD G2 G1 S

G2/M: 22.794% G2/M: 53.217% G2/M: 66.366% G2/M: 79.795%

AZD2281 induces apoptosis

MPNST 724 96 hour treatment 2.5uM AZD control 5.0uM AZD 10.0uM AZD

8% total apoptosis 30% total apoptosis 35% total apoptosis 42% total apoptosis

Effect on tumor growth

MPNST Xenograft: Subcutaneous Injection (16 mice)

Tumors grow to 5mm

Intraperitoneal injection

Vehicle (8 mice)

PBS+10%DMSO+10%HPCB

Treatment (8 mice)

50mg/kg/day AZD2281

Sacrifice mice when vehicle group reaches 1500mm 3 , measure tumor volume and weight, preserve tissue

AZD2281 abrogates tumor growth

p= 0.0002

* *

p= 0.0002

*

p= 0.0002

Conclusions

• MPNST cells are relatively sensitive to PARP inhibition

in vitro

– Decrease in cell proliferation – G2/ M cell cycle arrest – Enhanced apoptosis • Anti-tumor effect of PARPi

in vivo

– Cytostatic effect on tumor proliferation – Role of PARPi in combination with chemotherapy preclinical/clinical trials?

Acknowledgements

PARP Function and Inhibition

Histone- coated strip wells

PARP Activity Assay

AZD2281 ABT888 BSI 201

-Add PARPi of interest -Add cell lysate -Add PARP cocktail, incubate Read Absorbance at 450nm + Add TACS Sapphire Add HCl Biotinylated NAD:

NAD

Strepavidin HRP:

724

Complex MPNST Karyotype

The chromosome # in this cell line ranged from 47-91 , the modal chromosome # being 58

DNA damage PCR array Results

Name

Upregulated BRCA1 CCNO 37kDa EXO1 FEN1 Β-Actin LIG1 MSH2 NEIL3 PARP1 PARP 1 POLD3 RAD51 B-Actin RAD54L RPA3 XRCC2

Relative Fold Change to NSC

724 ST88 462 9.7( ± 3.2) 10.3( ± 4.8) 29.9( ± 8.7) 43.5( ± 24.4) 18.0( ± 6.1) 161.6( ± 86.7) 30.7( ± 5.2) 7.8( ± 3.6) 19.0( ± 6.8) 5.4( ± 0.6) 10.9( ±5.7) 6.6( ± 3.6) 8.7( ± 2.8) 11.0( ± 7.2) 29.7( ± 12.3) 5.5( ± 0.3) 24.5( ± 5.5) 32.2( ± 5.8) 13.1( ± 4.3) 24.6( ± 5.5) 5.3( ± 0.7) 13.7( ±10.1) 12.1( ± 6.1) 6.8( ± 2.7) 8.5( ± 4.1) 30.4( ± 7.6) 6.1( ± 0.5) 37.8( ± 6.41) 43.9( ± 10.9) 12.6( ± 3.6) 26.6( ± 9.9) 20.4( ± 3.3) 20.3( ±17.5) 12.6( ± 6.4) 6.5( ± 2.1) 15.6( ± 4.1) 38.4( ± 11.8) 5.5( ± 0.1) 30.9( ± 3.4)