PD Evaluations and Pathway Analysis of 2 PARP Inhibitors: ABT-888 vs. BSI201 J Ji & M Lee 11-12-2010 1.

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PD Evaluations and Pathway Analysis of 2 PARP Inhibitors: ABT-888 vs. BSI201 J Ji & M Lee

Two PARP Inhibitors: ABT-888 vs. BSI201 2

Lab Questions Is there difference in PD response? Does agent induce  h2AX? Explore genome expression through Next-gen Seq for drug targets Transcriptome/Pathway Analysis for MOA 3

PARP Inhibitions in Treated MX-1 and PBMC  M

 H2AX in MX-1 Treated with ABT888 2h 4h 6h 24h uM 0.8uM 10uM 5

 H2AX in MX-1 Treated with BSI uM 0.8uM 10uM 2h 4h 6h 24h 6

 H2AX Comparison No Drug 0.8 uM 2h 10 uM 24h ABT-888 BSI201 7

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 8

Summary of Sequence Reads and BWA Mapping SampleTotalMappedProperly Mapped MX1_control (95.11%) (82.04%) MX1_ABT_ (90.50%) (79.22%) MX1_BSI_ (91.74%) (78.42%) 1 lane of 76 base pair-end Mapping Pipeline Assemble bam files Hg18 reference sequence mRNA refseq EST Combination of any two exons 9

The Number of Genes that Showed Differential Expression between Control and Treated Cells TreatmentNumber 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 MX1_BSI_4h

Top 100 Differentially Expressed Genes Down-regulation Up-regulation ABT BSI 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 11

Telomere Elongation Controlled by Tankyrase 1 (PARP5) PINX1 is PIN2/TRF1 interacting, telomerase inhibitor 1 H. Seimiya, British Journal of Cancer (2006) 94, 341 –

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.  H2AX 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. 13

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 14