PD Evaluations and Pathway Analysis of 2 PARP Inhibitors: ABT-888 vs. BSI201

J Ji & M Lee

11-12-2010

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ONH2

NH

N

NH

CH3

Two PARP Inhibitors:ABT-888 vs. BSI201

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Lab Questions

• Is there difference in PD response?• Does agent induce gh2AX?• Explore genome expression through Next-gen Seq

for drug targets• Transcriptome/Pathway Analysis for MOA

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PARP Inhibitions in Treated MX-1 and PBMC

1 2 3 4 5 6 7 80

100

200

300

MX-1

ABT

BSI

Rel

ativ

e P

AR

Lev

els

(%)

0 .1 .2 .4 .8 1.6 10 20 mM

0 0.21 0.8 100

50

100

150

PBMC

ABT

BSI

Rela

tive

PAR

Leve

ls (%

)

gH2AX in MX-1 Treated with ABT8882h 4h 6h 24h

0

0.21uM

0.8uM

10uM

5

gH2AX in MX-1 Treated with BSI201

0

0.21uM

0.8uM

10uM

2h 4h 6h 24h

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gH2AX Comparison

No Drug

0.8 uM

2h

10 uM

24h

ABT-888 BSI201

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Rational for Next-generation Sequencing Analysis

Next-gen sequencing technology provides a high-throughput approach to the analysis of transcriptome with a single-nucleotide resolution.

(1) To identify genes with differential expression following PARP inhibitor treatment

(2) To identify alternative splicing forms altered by PARP inhibitor treatment

(3) To identify background mutations and translocations

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Summary of Sequence Reads and BWA Mapping

Sample Total Mapped Properly Mapped

MX1_control 41062746 39053394 (95.11%) 33686773 (82.04%)

MX1_ABT_4 49218414 44543496 (90.50%) 38990313 (79.22%)

MX1_BSI_4 45805500 42020160 (91.74%) 35918553 (78.42%)

1 lane of 76 base pair-end

Mapping Pipeline

Assemble bam files

Hg18 reference sequencemRNA refseqESTCombination of any two exons

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The Number of Genes that Showed Differential Expression between Control and Treated Cells

Treatment Number of genes showing down-regulation in treated

cells (FDR=0.1)

Number of genes showing up-regulation in treated cells

(FDR=0.1)

MX1_ABT_4h 9509 2289

MX1_BSI_4h 9531 1694

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Top 100 Differentially Expressed Genes

Down-regulation

Up-regulation

ABT BSI

95 5 95

81 19 81

telomere maintenance: DKC1 PINX1

rRNA processing:DDX56 DKC1 RPS19 WDR3

retinoic acid receptor binding: RING1 ZBTB22

cell redox homeostasis:RAC3 STMN3

transcriptional repressor: CREBZF PURB

heart development:ERBB3 NOTCH1 PKD1 SALL4

centriosome: PCNT TOP2A

chromosome organization:BRCA2 SMC4

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Telomere Elongation Controlled by Tankyrase 1 (PARP5)

PINX1 is PIN2/TRF1 interacting, telomerase inhibitor 1

H. Seimiya, British Journal of Cancer (2006) 94, 341 – 34512

Summary

• PARP inhibition was detected in cells treated with ABT888, but not BSI201 using MX-1 as tumor model and PBMC as surrogate. PAR reduction by ABT-888 was dose dependent, but not BSI201.

• gH2AX was increased post-dose for both agents, more induction with BSI201 in MX-1.

• Next-gen Seq & Gene Ontology analysis showed genes in different pathways were repressed or induced by ABT-888 and BSI201. BSI201 specifically suppresses genes in telomere pathway, suggesting PARP5/6 as potential targets, whereas ABT-888 induces genes in chromosome organization, supporting PARP1/2 as targets.

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Future Direction

• AACR Abstract Submission (due Nov 15, 2010) and prepare manuscript for submission to PNAS

• Confirm Pathway Analysis: 24h vs. 4 h and include 3 PARP Inhibitors: ABT-888; BSI201; AZD2281

• Validate Candidates with real-time qPCR– Dose Curve (0, 0.2, 0.8, 1, 2, 10 uM)– Time Course (0, 2, 4, 7, 24h)– More Cancer Lines: MCF-7, PC-3, Ovarian…

• Xenograft Exp to Verify in vitro data

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