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

PD Evaluations and Pathway Analysis of 2 PARP Inhibitors: ABT-888 vs. BSI201. J Ji & M Lee 11-12-2010. Two PARP Inhibitors: ABT-888 vs. BSI201. Lab Questions. Is there difference in PD response? Does agent induce g h2AX?

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

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

  2. Two PARP Inhibitors: ABT-888 vs. BSI201

  3. 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

  4. PARP Inhibitions in Treated MX-1 and PBMC 0 .1 .2 .4 .8 1.6 10 20 mM

  5. gH2AX in MX-1 Treated with ABT888 2h 4h 6h 24h 0 0.21uM 0.8uM 10uM

  6. gH2AX in MX-1 Treated with BSI201 2h 4h 6h 24h 0 0.21uM 0.8uM 10uM

  7. gH2AX Comparison ABT-888 BSI201 No Drug 0.8 uM 2h 10 uM 24h

  8. 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

  9. Summary of Sequence Reads and BWA Mapping Hg18 reference sequence mRNA refseq EST Combination of any two exons Mapping Pipeline 1 lane of 76 base pair-end Assemble bam files

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

  11. Top 100 Differentially Expressed Genes BSI ABT telomere maintenance: DKC1 PINX1 rRNA processing: DDX56 DKC1 RPS19 WDR3 retinoic acid receptor binding: RING1 ZBTB22 cell redox homeostasis: RAC3 STMN3 95 5 95 Down-regulation transcriptional repressor: CREBZF PURB heart development: ERBB3 NOTCH1 PKD1 SALL4 centriosome: PCNT TOP2A chromosome organization: BRCA2 SMC4 81 19 81 Up-regulation

  12. Telomere Elongation Controlled by Tankyrase 1 (PARP5) PINX1 is PIN2/TRF1 interacting, telomerase inhibitor 1 H. Seimiya, British Journal of Cancer (2006) 94, 341 – 345

  13. 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.

  14. 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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