1. 2 E Conférence Québécoise Sur La Résistance Thérapeutique du Cancer Q-CROC Montréal, November 5-6, 2010

2. Potential Conflict of Interest Dr. Thierry Muanza –None

3. Radiotherapy  Along with surgery and chemotherapy, radiotherapy is a mainstay of cancer treatment  75% cancer patients in the course of their disease will undergo radiotherapy

4. Radiobiology  IR local release of large amount of energy  ~ 33eV dissipated / ionizing event, enough to break strong chemical bond  energy associated C=C bond is 4.9 eV  Types:  Electromagnetic  particulate

5. IR: mechanism of action.

6. Radiation Induced DNA Damage http://www.radiation-scott.org/radsource

7. Chromosomal Damage ApoptosisReproductive death Necrosis

8. Radiation Induced DNA Damage ATM G1 arrest Repair G2 arrest G1 arrest Apoptosis G2 arrest pH2XA P53 BAX Cytochrome c Apaf1 Caspase 9 Caspase 6 Caspase 3 Apoptosis P53 Cycline B N Cycline-B-p34 cdc2 p21 WAF1 / CIPI cdK /Cycline D Cycline E pRb E2F pRb E2F Smac/ Diablo Survivin

9. DNA Repair Mechanisms ATM DSB Rad51 /Rad52 Rad52/MRE11/NBSI BRAC1/BRAC2 Completion of DSB repair DNA-PKcs / Ku80/70 MRE11/Rad51/NBSI DNA ligase IV, XRCC4 RNA Pol II or XPC XPD / XPB ERCC1 / XPG Completion of repair DNA Pol  / ligase DNA Pol  / DNA ligase III XRCC1 BER NHEJ HR pH2aX NER SSB

10. Cell Survival Curves

11. CA: RT sensitivity

12. Cell cycle: RT sensitivity

13. Tumor Oxygenation

14. Re-oxygenation

15. Effect of Oxygen

16. RT resistance Ma et al. JCO 21, 2003

17. Radiosensitizers

19.  Nonhypoxic  Halogenated pyrimidines  DNA  Cycling tissues  Hypoxic  Free radical process (fixed)  Misonidazole (toxicity)  Overgaard metaanalyisis: 4.6% LC, 2.8% OS  Hypoxic cytotoxins  Bioreductive Rx  Tirapazamine (nitroxide)  Mitomycin C

20. Radiosensitizers

22. Cell Proliferation Assay (MTT) MDA-MB-468 cells Heravi, Muanza et al. ACD 20, 2009

23. Cell Proliferation Assay (MTT) **: P<0.01

24. Colony Forming Assay Radiation Dose (Gy)DER 246246 1.6 2.2 2.9

27. γH2AX Immunofluorescent Staining Control 1 Gy 0.5 Gy

28. γH2AX Immunofluorescent Staining Control ZRBA1 18uM ZRBA1 9uM

29. γH2AX Immunofluorescent Staining Control 0.5 Gy ZRBA1 9uM0.5 Gy and ZRBA1 9uM

30. γH2AX Immunofluorescent Staining Control 1 Gy ZRBA1 9uM 1 Gy and ZRBA1 9uM

31. γH2AX Immunofluorescent Staining

32. FACS Analysis 1 hrs post treatment 24 hrs post treatment

33. Comet Assay

34. Control ZRBA1 Radiation ZRBA1 and radiation

35. EGFR Inhibitory Activity (ZRBA1)

36. EGFR Signaling Pathway TK TGF RAS RAF ERK1/2 BAD Apoptosis Caspase 9 Caspase 3 Survival AKT EGFR P Serine136 Serine112 P PI3K P   Over-expression of EGFR associates with activation of the AKT pathway. P HER2 MEK

37. Conclusions  Significant cell killing in cells exposed to combination of ionizing radiation with ZRBA1.  Higher G2M arrest in cells exposed to the combined treatment.  The most effective schedule for this combination is administration of drug before and / or concurrent with radiation.  ZRBA1 potentiates the effect of radiation in MDA-MB-468 cells.  The combination of IR and ZRBA1 induces the formation of γH2AX foci.

38. Future Plans  Better understanding of the molecular mechanism induced by this novel therapeutic approach.  Evaluate the status of DNA repair proteins.  Evaluate the expression levels of antiapoptotic and proapoptotic proteins.  Investigation for other possible modes of cell death.  In-vivo correlative studies.

39. Cancer Stem Cells  Self-renewing cell  Pluripotent cell: recapitulate its tumor in SCID mice

40. CSC: mechanisms of resistance

41. CSC: RT resistance  CSC enrichment  Chk1/Chk2 inhibitor S. Bao Nature 444, 2006

42. CSC: RT resistance  apoptosisCell cycle arrest

43. CSC: DNA damage repair kinetics

44. Future directions  Clinical models for identification of RT resistance  Rectal cancer  CNS tumors  Zevalin resistant lymphoma

45. Acknowledgments  Dr. Thierry Muanza - Mitra Heravi - Lillian Lee - Azusa Maeda - Ava Schlisser  Dr. Danuta Radzioch  Dr. Bertrand Jean-Claude (Cancer Drug Research Laboratory) - Dr. Zakaria Rachid - Margarita Todorova

46. Thank You!