Bacterial Tumor Therapy against Melanoma

1. Bacterial Tumor Therapy against Melanoma

2. Steps in melanoma progression From: „Cellular and molecular biology of human melanoma.“, Satyamoorthy et al. Cancer Biol Ther. 2002 Jan-Feb;1(1):14-7

3. Ras/Raf-Singnalling Rapp et al. From: Rapp et al. (2003) Adv Enzyme Regul. 43:183

4. RAF-family • 3 members: A-, B-, C-Raf • Constitutive low signals lead to cell proliferation and very high signals to cell cycle arrest • High levels induce p21 expression • Raf oncogene cooperates with Myc • Inactivates pro-apoptotic Bcl2 family proteins • Interacts directly with proteins like pyruvate kinase

5. Projects • Transgenic BRafV600E mouse to determine further steps in melanoma progression, which might be useful to develop a new therapy • Bacterial tumor therapy to use bacteria to deliver a system to the tumor, that converts a prodrug into a drug

6. Transgenic BRafV600E-mouse The expression of the BRafV600E should be tissue-specific or inducible, because the strong expression of an oncogene would probably interfere with the normal embryonal development

7. Cre/loxP system From: „ Inducible Gene Targeting in Mice Using the Cre/lox System.“ Sauer B, A Companion to Methods in Enzymology 14, 381-392 (1998)

8. Cloning strategy for pCMV5-NeoLoxP-BrafV600E-Myc In vitro annealing PCR XbaI overhang XbaI XbaI pCMV5 Killed XbaI site XbaI site Ligation pCMV5 pCMV5 Myc-tag Ligation CMV promotor Floxed neo B-Raf*

9. „Tet-On“-System From: Zhou, Z. et al. (2002), Cell and Developmental Biology, 13 (121-128)

10. pBI5-BRafV600E-Myc Ptet-bi1: bidirectional Promotor BRafV600E-Myc Luciferase

11. Bacterial tumor therapy • Attenuated intracellular bacteria like Salmonella typhimurium or Listeria monocytogenes locate tumors • Recombinant bacteria that locate tumors, can be used to deliver a system to the tumor, that converts a prodrug into a drug

12. Bacterial tumor therapy in melanoma Injection of recombinant Listeria monocytogenes Subcutaneous injection of B78-D14 cells Injection of the prodrug • Homogenization of the tumor • CFU • drug/prodrug concentration

13. Bacterial tumor therapyInfection of B16-cell line with Listeria monocytogenes aroA/B • Listeria monocytogenesaroA/B strain can invade B16 cell line, although it is highly attenuated • Infection efficiency is not very high Mutagenesis might help to get a clone that gives a higher infection efficiency?

14. Mutagenesis • Mutagenesis of Listeria with EMS • determination of the mutation rate by plating the bacteria on rifampicin plates • selection of the bacteria, that still are able to invade the cells by infection of the B16 cell-line Mutagenesis 3 x Infection

15. Infection with mutated Listeria monocytogenes

16. B78-D14 4T1 3T6 J774

17. In-vivo infection with L.monocytogenes  trpS aroA/B

18. Conclusion BRafV600E transgenic mouse might give us the possibillity to determine further steps in melanoma progression, that might be useful for developing a new therapy Mutagenesis might give us a possibility to generate a Listeria strain that is better adapted to the melanoma cells than the original strain