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From Chinese Plants to Western Medicine: The Story of Antitumor Camptothecins

From Chinese Plants to Western Medicine: The Story of Antitumor Camptothecins. Leroy F. Liu Department of Pharmacology University of Medicine and Dentistry of New Jersey (UMDNJ)- Robert Wood Johnson Medical School The Cancer Institute of New Jersey. Xenograft line. Type. Histology a.

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From Chinese Plants to Western Medicine: The Story of Antitumor Camptothecins

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  1. From Chinese Plants to Western Medicine: The Story of Antitumor Camptothecins Leroy F. Liu Department of Pharmacology University of Medicine and Dentistry of New Jersey (UMDNJ)- Robert Wood Johnson Medical School The Cancer Institute of New Jersey

  2. Xenograft line Type Histologya ADRb 5-FUra MTX CYT ALK VCR VBL MCCNU BCNU 9-NC 9-AC 10,11-MDC Case Colon P 0c 0 0 0 0 0 PR CR CR 0 0 0 SW48 Colon P 0 0 0 0 0 0 CR 0 0 0 SQU Colon M 0 0 0 0 0 0 CR 0 0 0 BRE Stomach M DOY Lung UN CR 0 0 0 0 0 0 0 0 0 CR HAR Lung SC 0 0 0 pi 0 0 PR gi 0 0 CR SPA Lung AD CR DIL Lung EP PR CR gi CLO pi 0 gi gi ID gi Breast PR PR 0 0 0 0 MUR ID CR CR 0 0 0 0 0 0 0 0 Breast LAN Ovary UN CR Malignant melanoma BRO 0 0 0 pi 0 0 0 0 CR PR Malignant melanoma SCH 0 0 pi pi pi pi 0 0 gi Giovanella et al. Cancer Res. 51:3052-3055, 1991 O O N N OH N N O O HO HO O O- Na+ camptothecin lactone camptothecin sodium salt • inactive • binds HSA Exceptional Antitumor activity of camptothecins in human cancer xenografts

  3. Camptothecin Derivatives in Development Pre-registered Phase II Registered Gimatecan Novartis/Sigma-Tau Rubitecan SuperGen Pancreas, p.o Lurtotecan OSI Topotecan (GSK) 1996 Ovary 2nd line, SCLC (USA) BNP-1350 Karenitecin Bionumerik Diflomotecan, BN-80915 Roche/Beaufour Phase III Irinotecan 1994 Aventis, Daiichi, Pharmacia Colon 1st line Exatecan Daiichi CKD-602 J&J/C-K Dang Pre-clinical BN-80927 Roche/Beaufour Plus conjugates; 2 are in Phase I (Daiichi, Cell Therapeutics) 2 in Phase II (Bayer, Enzon)

  4. Why Do Camptothecins Exhibit Exceptional Antitumor Activity? • Camptothecins target DNA topoisomerase I • Exquisite S phase-specific cytotoxicity: Replication fork collisions • Defective repair of TOP1 cleavage complexes in tumors: ISG15 interference with TOP1 degradation

  5. 1. DNA replication elongation DNA replication + 2. Transcription elongation + + + Topoisomerase I Solves Topological Problems of DNA catalysis covalent intermediate Tyr 5’

  6. Camptothecin traps topoisomerase I-DNA covalent intermediate: a reversible TOP1 cleavage complex O N A B C A D N E O 20 SDS or alkali + OH O B Camptothecin SDS or alkali +CPT C OH strand passing D TOP1 is elevated in many human tumors!

  7. Why Do Camptothecins Exhibit Exceptional Antitumor Activity? • Camptothecins target DNA topoisomerase I • Exquisite S phase-specific cytotoxicity: Replication fork collisions • Defective repair of TOP1 cleavage complexes in tumors: ISG15 interference with TOP1 degradation

  8. + CPT S phase-specific killing: a replication fork collision model OH replication fork arrest, fork breakage, cleaved complex ATM/ATR Chk1 53BP1 P53 NFkB RPA Cell Death/ Cell cycle arrest

  9. Why Do Camptothecins Exhibit Exceptional Antitumor Activity? • Camptothecins target DNA topoisomerase I • Exquisite S phase-specific cytotoxicity: Replication fork collisions • Defective repair of TOP1 cleavage complexes in tumors: ISG15 interference with TOP1 degradation

  10. Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Defective Repair of TOP1-DNA Covalent Adducts in Most Tumor Cells RNA polymerase II +CPT proteasomal degradation of TOP1 & RNA pol II LS TOP1 down-regulation • In tissue culture cells • In mouse models • In patients receiving • topotecan treatment repair Desai, S. D., Zhang, H., Rodriguez-Bauman, A., Yang, J.-M., Wu, X., Gounder, M. K., Rubin, E. H. and Liu, L. F. “Transcription-Dependent Degradation of Topoisomerase I-DNA Covalent Complexes” Mol. Cell. Biol. 23:2341-2350 (2003). transcription restart

  11. 14.3 kDa (UCRP) 6.2 kDa (ubiquitin) Breast cancer cells BT474 ZR-75-1 80 BT474 60 40 % TOP1 degradation 14.3 kDa (UCRP) 20 ZR-75-1 0 0 2 4 6 6.2 kDa (ubiquitin) Time (hr) Elevated levels of ISG15 (UCRP) in tumor cells: inverse correlation with Top1 degradation T-antigen-transformed Colon cancer cells human lung cells HCT116 KM12 HT29

  12. Ubiquitin-like protein ISG15 is elevated in tumor biopsies -actin

  13. * APG12 * FAT10 * URM1 * SUMO1/2/3 Ubiquitin M L V L R L R GG 68 F M N L R L R G G ISG15 149 SUMO1 E V Y Q E Q T G G 89 Nedd8 M L V L A L R G G 68 (Interferon-Stimulated Gene 15): A ubiquitin-like protein (UBL) ISG15 * RUB1 * NEDD8 Bacterial infection Virus infection Type I IFN LPS Tumorigenesis ISG15 UBE1L (E1) UbcH8 (E2) E3 ? Protein Protein-ISG15 UBP43

  14. siRNA-Mediated Down-Regulation of ISG15 or UbcH8 Restores Top1 Degradation TOP1 TOP1 TOP1 tubulin tubulin tubulin Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Control siRNA + CPT 0 2 4 6 hr RNA polymerase II +CPT ISG15 siRNA tumorigenesis + CPT 0 2 4 6 hr ISG15 UbcH8 siRNA + CPT 0 2 4 6 hr degraded TOP1 & RNA pol II LS ISGylation interferes with TOP1 degradation!

  15. How does ISG15 interfere with TOP1 down-regulation in tumor cells? 1. ISGylation specifically interferes with TOP1 degradation? 2. ISGylation interferes with the ubiquitin/26S proteasome pathway ?

  16. Tumorigenesis ISGylation ISG15 UBE1L (E1) UbcH8 (E2) E3 ub 26S proteasome + E1/E2/E3 substrate substrate ubiquitination Desai et. al. Cancer Res. 2006. ISGylation interferes with the Ub/26S proteasome pathway ISG15 or UbcH8 siRNA restores protein polyubiquitination and degradation. protein degradation

  17. Overexpression of ISG15 intumor cells inhibits TOP1 degradation through interference with the ub/26S proteasome pathway +CPT Tumorigenesis ISG15 Ub/26S proteasome

  18. +CPT ISG15 Tumorigenesis Ub/26S proteasome repair Elevated ISG15 Pathway in Tumors Confers CPT Sensitivity

  19. Camptothecins: problems in the clinic O N A B C Not as efficacious in humans as in mice D N E O 20 OH O camptothecin • Possible reasons: • Reduced potency due to opening of the lactone ring • Reduced potency due to drug efflux (e.g. substrates for BCRP) • Interpatient variability due to drug metabolism (e.g. prodrug activation and glucuronidation) Our solution: Target-based approach: To explore new structural classes of TOP1-targeting drugs

  20. A New Class of TOP1-Directed Anticancer Drugs CH2 N(CH3)2 N N O O H O O O H H C C O O N N 3 3 O O 10 10 N N N N C C H H 3 3 H H C C O O N N O O 3 3 1 1 O O C C H H 3 3 Topotecan ARC-111 O O H H O O SKNEP Aplastic Wilms Tumor 0.1 0.1 0 2 4 6 8 10 12 0 2 4 6 8 10 12 10 control ARC-111 (2 mg/kg) 1 3 TUMOR VOLUME (CM3) TUMOR VOLUME (CM3) 1 0.1 0 2 4 6 8 10 12 0.1 0 2 4 6 8 10 12 WEEKS WEEKS control Topotecan (2 mg/kg) N O TUMOR VOLUME (CM3) TUMOR VOLUME (CM3) H C O 3 O H C O N O 3 WEEKS WEEKS N ARC-111 • chemically stable • high potency • Minimal HSA binding • not a Pgp or BCRP substrate SZ-I • Model-directed synthesis • Most active Top1 drug SZ-I-181; IC50 0.15 nM

  21. Summary • The plant alkaloid camptothecin kill tumor cells by trapping TOP1-DNA covalent adducts which leads to S phase-specific cytotoxicity (replication collisions). • Tumor cells are hypersensitive to camptothecin due to defect in repair of TOP1-DNA covalent adducts (ISG15 overexpression). • Mechanism-based development leads to identification of a new class of TOP1 poisons, devoid of the labile lactone ring.

  22. Collaborators: Dr. Edmond LaVoie (Rutgers) Dr. Eric Rubin (CINJ) The Liu Lab-2005 Larry Wood Shyamal Desai Lisa lyu

  23. ISG15: A link between tumorigenesis and drug resistance tumorigenesis virus infection Type I IFN ISG15 UBE1L (E1) UbcH8 (E2) E3 bacterial infection LPS ubiquitin/26S proteasome pathway Protein Protein -ISG15 UBP43 immune evasion drug sensitivity

  24. Viral Infection Enahnces Class I Antigen Presentation: An proteasome/immunoproteasome switch viral infection IFN viral proteins ISG15 LMP-2, LMP-7, MECL-1 26S proteasome immunoproteasome antigenic peptides + MHC-I class I antigen presentation

  25. Tumorigenesis and Immune Evasion? tumorigenesis ISG15 UBE1L (E1) UbcH8 (E2) E3 ubiquitin/26S proteasome pathway Protein Protein -ISG15 UBP43 class I antigen presentation immune evasion

  26. Many Questions Remain ! • How is the ISG15 pathway turned on during tumorigenesis? • How does the ISG15 pathway interfere with the ubiquitin/26S proteasome pathway? • Why is the ISG15 pathway turned on during tumorigenesis?

  27. RNA polymerase II +CPT tumorigenesis Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub ISG15 degraded TOP1 & RNA pol II LS Overexpression of ISG15 inhibits TOP1 degradation

  28. Interferon sensitization of resistant tumors to CPT • Use of type I interferon to induce the ISG15 pathway in certain CPT-resistant (low ISG15) tumors. • Synergy expected with interferon/CPT combination IFN IFN a ISGylation control Defective TOP1 degradation CPT-11 CPT-11 + IFN a CPT sensitivity Kobayashi I. and Maemura M. (1996) Anticancer Res.16, 2677-2680

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