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

Potential Conflict of Interest Dr. Thierry Muanza –None

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

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

IR: mechanism of action.

Radiation Induced DNA Damage

Chromosomal Damage ApoptosisReproductive death Necrosis

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

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

Cell Survival Curves

CA: RT sensitivity

Cell cycle: RT sensitivity

Tumor Oxygenation

Re-oxygenation

Effect of Oxygen

RT resistance Ma et al. JCO 21, 2003

Radiosensitizers

 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

Radiosensitizers

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

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

Colony Forming Assay Radiation Dose (Gy)DER

γH2AX Immunofluorescent Staining Control 1 Gy 0.5 Gy

γH2AX Immunofluorescent Staining Control ZRBA1 18uM ZRBA1 9uM

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

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

γH2AX Immunofluorescent Staining

FACS Analysis 1 hrs post treatment 24 hrs post treatment

Comet Assay

Control ZRBA1 Radiation ZRBA1 and radiation

EGFR Inhibitory Activity (ZRBA1)

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

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.

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.

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

CSC: mechanisms of resistance

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

CSC: RT resistance  apoptosisCell cycle arrest

CSC: DNA damage repair kinetics

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

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

Thank You!