Proceedings of the 52nd Annual ASTRO Meeting S643

Conclusions: These results implicate a synergistic tumor control effect of RT and PSA Listeria vaccine in mice PC model. At thecellular level, this efficacy was further demonstrated by INF-y production and PSA-specific CTL activation in both spleen andtumor. A concurrent therapy of Listeria PSA vaccination and RT may provide an alternative approach for intermediate, highrisk and metastatic prostate cancer patients. Currently, our endeavors are directed towards verifying this synergy in transgenicPSA-expressing mice.

Author Disclosure: R. Hannan, None; P. Cohen, None; L. Lu, None; H. Zhang, None; S. Saha, None; R. Kabarriti, None; A. Alfieri,None; M. Garg, None; S. Kalnicki, None; C. Guha, None.

2984 MEK/ERK Inhibition by U0126 Radiosensitizes Rhabdomyosarcoma Cells In Vitro and In Vivo

F. Marampon1, G. Gravina1, P. Bonfili1, M. Di Staso1, C. Fardella1, L. Polidoro1, C. Festuccia1, R. G. Pestell2, V. Tombolini1,

B. M. Zani1

1University of L’Aquila, 67100, Italy, 2Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA

Purpose/Objective(s): Rhabdomyosarcoma (RMS) is a childhood solid tumor, resulting from dysregulation of the skeletal myo-genesis program. Combined-modality treatment has improved the outcome and radiosensitizers are used to enhance the radiother-apeutic (RT) efficiency. The problem of radioresistance and molecular mechanisms by which RMS cells overcome radioresistantremains to be elucidated. Several evidences demonstrate that increased radioresistance is associated with the expression of acti-vated oncogenes, including Ras and c-Myc and that the expression of mutant Ras increases cellular radioresistance. Mutated formsof Ras are found in 30% of human cancers including RMS. Mutations produce a constitutively active Ras protein relaying uncon-trolled signals such as the pathological c-Myc protein accumulation. We reported that the MEK/ERK inhibition, counteracting thec-Myc protein accumulation, blocks the RMS transformed phenotype in vitro and in vivo. This study was designed to examinewhether the MEK/ERK pathway affects intrinsic radiosensitivity in RMS cancer cells.

Materials/Methods: Human RMS, RD and TE671 cell lines were used. The cells were treated with the specific MEK/ERK in-hibitor U0126 (10uM), in combination with RT (4 Gy). ERK, c-Myc-, Ki67-, CD31-endothelial-marker-protein level and activa-tion were visualized by immunobiochemical and immunohistochemical analysis. Radiosensitivity was evaluated in vitro bycolony-forming essays and in vivo by xenograft models. The phosphorylation of histone H2AX was used as marker for irradia-tion-induced DNA double-strand break (dsDNA). For the c-Myc silencing, RMS cell lines were stable infected with a Tetracy-cline-inducible-shRNA-c-Myc vector.

Results: U0126 conferred greater radiosensitivity on the RMS tumor cells effecting a significant decrease in colony formationand increase in dsDNA damage, compared with radiation alone. In vivo, the addition of U0126 to radiation resulted in a tumor-growth-delay enhancement ratio of 3.5 over radiation alone and extended survival time. Combined treatment reduced tumorvolume, blood vessel density and increased the phosphorylation of histone H2AX. Mechanistically, U0126 was shown to radio-sensitize down-regulating the c-Myc protein. The radiosensitivity was increased by the c-Myc silencing in in vitro and in vivoessays.

Conclusions: RT is commonly used to treat RMS disease. Despite attempts at dose escalation outcomes remain poor. U0126, a se-lective inhibitor of RAS/MEK/ERK pathway, radiosensitized RD and TE671 cell lines both in vitro and in vivo counteracting theabnormal accumulation of c-Myc protein . Thus, U0126 may be investigated for a signal transduction-based targeting of the MEK/ERK/c-Myc oncogenic axis as a therapeutic strategy for radiosensibilization in RMS.

Author Disclosure: F. Marampon, None; G. Gravina, None; P. Bonfili, None; M. Di Staso, None; C. Fardella, None; L. Polidoro,None; C. Festuccia, None; R.G. Pestell, None; V. Tombolini, None; B.M. Zani, None.

2985 Targeting Prosurvival Bcl-2 Family Members Increases Radiation Sensitivity in Breast Cancer Cells

H. Wu, D. S. Schiff, S. Goyal, B. G. Haffty

Department of Radiation Oncology, Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, NewBrunswick, NJ

Purpose/Objective(s): In previous studies we have shown that Bcl-2 expression was associated with an increased risk of localrecurrence in patients with early stage breast cancer (Yang Q, Breast Cancer Res Treat, 2009). In addition, Bcl-2-overexpressinglymphoma cell lines are resistant to radiation-induced apoptosis (Mirkovic N, Oncogene, 1997). The recent availability of drugsthat target Bcl-2 makes the strategy of Bcl-2 targeted therapy in combination with radiation appealing. The purpose of this studyis to explore an approach to circumventing radiation resistance by targeting prosurvival Bcl-2 family proteins in breast cancercells.

Materials/Methods: The breast cancer cell lines MCF-7, T47D and MDA-MB231 were used. To silence the anti-apoptotic Bcl-2protein and its related family member Mcl-1, siRNAs were adopted. In addition, a small molecule inhibitor of Bcl-2, ABT 737 (Sup-plied by Abbott Laboratories), was used to target anti-apoptotic Bcl-2 family proteins. In order to assess Bcl-2 related cell death, Bakand cleaved PARP, an apoptosis indicator, were determined by Western blot analysis. Cell viability was measured by MTT assay.

Results: Cleaved PARP in Bcl-2 siRNA treated MCF-7 cells increased compared to non-targeting siRNA treated cells 72 hours after 8Gy of radiation, indicating targeting Bcl-2 alone increased apoptotic cell death. Despite evidence of increased apoptosis, cell viabilityshowed no significant difference between Bcl-2 siRNA and non-targeting siRNA treated MCF-7 cells at 2, 4, 6 and 8 Gy, p.0.05,indicating that targeting the Bcl-2 protein alone did not enhance radiation sensitivity. However, the combination of radiation (8Gy)and ABT 737 (variable doses) enhanced Bak and cleaved PARP at 24h, 48h and 72h. Further, the cell viability of the combinationof radiation (2Gy) and ABT 737 (5mM) significantly decreased 16.5%, p\0.01 and 26.5%, p\0.01, compared with radiation aloneor ABT 737 alone treated MCF-7 cells, respectively. Similar results were observed in T47D and MDA-MB231 cells. Finally, afterknocking down Mcl-1 by siRNA, cell viability went down in 14.5% (p\0.05) in Mcl-1 siRNA treated MCF-7 cells compared withnon-targeting siRNA treated cells at 5 mm of ABT 737. However, similar decreased percentage (15.6%) of cell viability also was de-tected after combination radiation (2Gy), siRNA and ABT 737 (5mM), suggesting, down-regulating Mcl-1 did not affect radiation sen-sitivity in MCF-7 cells.

S644 I. J. Radiation Oncology d Biology d Physics Volume 78, Number 3, Supplement, 2010

Conclusions: Targeting Bcl-2 prosurvival family members sensitizes breast cancer cells to radiation through activating Bak-ap-optotic pathway. These results support the combination of radiation and ABT 737 as a potential novel therapeutic strategy in breastcancer treatment.Supported by the Breast Cancer Research Foundation.

Author Disclosure: H. Wu, None; D.S. Schiff, None; S. Goyal, None; B.G. Haffty, None.

2986 Proton Therapy Targets Cancer Stem Cells in Treatment-resistant Non-small Cell Lung Cancer

J. Y. Chang, X. Zhang, O. Vassiliev, M. Gillin, R. Mohan

M. D. Anderson Cancer Center, Houston, TX

Purpose/Objective(s): Treatment resistance caused by cancer stem cells (CSCs) is a challenging clinical issue. Targeting CSCsmay improve cancer cure. The relative biological effectiveness (RBE) of protons is assumed to be close to that of photons at 1.1.However, the tissue- and cell-specific RBEs and the molecular mechanisms of proton therapy in treatment-resistant cancer cellssuch as CSCs are not well understood. We hypothesized that protons may be more effective than photons in eliminating CSCs,thereby reducing the probability of recurrence and metastasis.

Materials/Methods: We established 2 chemotherapy-resistant (CR) non-small cell lung cancer (NSCLC) cell lines (H460/CR andA549/CR) by repeatedly treating parental H460 and A549 cells with paclitaxel and 3 radiation-resistant (RR) NSCLC cell lines(H460/RR, A549/RR, and Seg-1/RR) by repeatedly irradiating parental cells to up to 60 Gy in 2-Gy fractions. Both CR andRR cell lines expressed higher percentages of ‘side population’ (SP) cells (i.e., those showing CSC phenotype) than did parentalcells (H460/RR, 15% ± 0.7%; H460/CR, 35% ± 1.1%; H460, 3% ± 0.4%; p\0.05). Enrichment was further enhanced after serialsorting and reanalysis (H460/RR, 35%; H460/CR, 52%) and confirmed by the tumorigenicity of the cells in nude mice after serialdilution. CR or RR CSCs and normal bronchial epithelial (NHBE) cells were then irradiated with the same doses (RBE) of protonsor photons and analyzed for growth, apoptosis, and invasion (metastatic potential). We further measured intracellular concentra-tions of reactive oxygen species (ROS), critical mediators of radiation-induced cell killing, by 2’-7’-dichlorofluorescein diacetatestaining and flow cytometry in parental cells and CSCs before and after photon or proton irradiation.

Results: At the same dose (RBE), protons killed significantly more than did photons (p\0.05), but this effect was not seen inNHBE cells. Protons induced significant increases in apoptosis compared with photon (p\0.05). Protons reduced metastatic po-tential in all cell lines tested, particularly in CSCs, in a dose-dependent manner; by contrast, photon-induced apoptosis was notdose-dependent and photons increased cellular invasion at lower doses (0.5 or 1 Gy). CSCs contained less than half the ROS levelsof the parental cancer cells or the NHBE cells. Notably, protons induced higher ROS levels than photons in CR/RR CSCs (3.3-foldincrease from protons vs. 2.1-fold increase from photons in H460/CR/SP cells), but protons and photons produced equivalent,smaller increases in ROS levels in NHBE cells.

Conclusions: Protons preferentially target CSCs and increase ROS levels in treatment-resistant CSCs to a greater extent than pho-tons for the same dose (RBE), but protons and photons produced equivalent effects in NHBE cells.

Author Disclosure: J.Y. Chang, None; X. Zhang, None; O. Vassiliev, None; M. Gillin, None; R. Mohan, None.

2987 Combining Integrin Inhibition (Cilengitide), EGFR Inhibition (Cetuximab) and Radiation in a Pancreatic

Cancer Model

C. Timke1,2, E. Fritz2, S. Schoelch3,2, F. Roeder1,2, A. Abdollahi1,2, J. Debus1, M. Koch3, S. Goodmann4, P. E. Huber1,2

1University of Heidelberg, Dept. Radiationoncology, Heidelberg, Germany, 2DKFZ German Cancer Research Centre,Heidelberg, Germany, 3University of Heidelberg Dept. Surgery, Heidelberg, Germany, 4Merck KG, Darmstadt, Germany

Purpose/Objective(s): Combination of targeted drugs is considered as one possibility for improving the therapeutic index in can-cer therapy. However, it is far from obvious which targeted drugs can favorably be combined. Combining radiotherapy with eitherthe EGFR antibody Cetuximab or the small molecule integrin inhibitor Cilengitide in dual combinations has shown promise inpreclinical and clinical settings. We analyzed the therapeutic potential of combining all three modalities in a human pancreatic can-cer model in vitro and in vivo.

Materials/Methods: Human dermal microvascular endothelial (HDMEC) and BXPC3 human pancreatic cancer cells were ex-posed to Cilengitide, Cetuximab and radiation alone and in dual and triple combinations with various concentrations and doses.In vitro, proliferation, migration/invasion, clonogenic and tube formation assays were performed and apoptosis was measuredby FACS. Genome wide transcriptomics (44k Agilent platform) was performed along with qRT-PCR and Western for protein anal-ysis. In vivo, in a s.c. BXPC3 model in balb c nude mice the effects of mono, dual and triple treatments with concurrent radiation(5x2Gy) were analyzed in 8 groups by tumor growth, histology and imaging using MRI, CT, and US.

Results: We found that in vitro and in vivo (e.g. tumor growth delay), the anti-endothelial and anti-tumor effects of the triple com-bination consisting of Cetuximab, Cilengitide and radiation was superior to dual combinations, while dual combinations were su-perior to monotherapies. The superiority was evident in cell proliferation, migration, tube formation, clonogenic survival, andapoptosis assays. Supra-additivity was found in isobologram analyses for the Cilengitide and Cetuximab combination in prolifer-ation and clonogenic assays as well as for reducing tumor growth in vivo. Exploring the underlying signaling mechanisms, wefound by transcriptomics in vitro that the Cilengitide drug target (integrinbetaV) is upregulated by radiation and Cetuximab, whichwas confirmed by IHC in vivo. Moreover, Cetuximab suppressed the Cilengitide and radiation induced upregulation of VEGF sig-naling. The strong antiangiogenic effects of the triple combination with a short period of transient vessel normalization in vivo wasevident in SMA/CD31 IHC along with corresponding perfusion analyses of contrast enhanced MRI/CT and US imaging.

Conclusions: Our data suggest that Cilengitide and Cetuximab can favorably be combined in dual combinations and in a triple combi-nation with radiotherapy in a human pancreatic cancer model. The combinatorial benefits can be explained in part in terms of the under-lying signaling mechanisms. Clinical translation for pancreatic carcinoma seems feasible due to the clinical availability of all modalities.

Author Disclosure: C. Timke, None; E. Fritz, None; S. Schoelch, None; F. Roeder, None; A. Abdollahi, None; J. Debus, None; M.Koch, None; S. Goodmann, at Merck KG, A. Employment; P.E. Huber, None.