- Email: [email protected]
NRF2 Is a Novel Oncogene and Biomarker of Therapeutic Resistance in Non-Small Cell Lung Cancer
S104
International Journal of Radiation Oncology Biology Physics
embryo fibroblasts (MEFs). The stress response to ionizing radiation YTR107 was assessed in wild-type and NPM-null MEFs and 6 NSCLC cell lines by quantifying pNPM1, gamma-H2AX, and Rad51 foci, neutral comet tail moment, and colony formation. In vivo efficacy of YTR107 radiosensitization was tested in a syngeneic xenograft model of tumor growth delay. NPM1 levels in a human-derived NSCLC tissue microarray (TMA) were determined by immunohistochemistry. Results: We observed that proliferating NPM1-null MEFs, but not wildtype MEFs, have high levels of gamma-H2AX foci, a characteristic of replication stress. In addition, NPM1-null MEFs exhibit increased radiation sensitivity via a defect in Rad51-mediated repair of DNA DSBs. We further determined that YTR107-mediated radiosensitization requires NPM1. The DNA damage response pathway is continuously activated in NSCLC due to constant replication stress. Because NPM1 expression minimizes replication stress, we further hypothesized that NSCLC tumors may have significant expression of NPM1. This was validated by analyzing a NSCLC TMA that demonstrated very low NPM1 expression in matched normal lung parenchyma but significantly increased NPM1 expression in Stage III NSCLC cores. We then observed that YTR107-mediated targeting of NPM1 resulted in radiosensitization in 6 NSCLC cell lines in vitro and in a syngeneic tumor model in vivo. Conclusions: These data demonstrate genetic evidence of NPM1 contribution to repair of radiation-induced DNA damage and suggest that NPM1 represents a potential therapeutic target for radiosensitization of NSCLC. Author Disclosure: C.A. Smith: None. K.R. Sekhar: Q. Patent/License Fee/Copyright; Co-inventor on the patent covering YTR107. M. Benamar: None. A. Venkateswaran: None. S. Sasi: None. N.R. Penthala: Q. Patent/License Fee/Copyright; Co-inventor on the patent covering YTR107. P.A. Crooks: Q. Patent/License Fee/Copyright; Co-inventor on the patent covering YTR107. S.R. Hann: None. L. Geng: None. T. Abbas: None. M.L. Freeman: Q. Patent/License Fee/Copyright; Co-inventor on the patent covering YTR107.
abrogated the effect of EGFR inhibition on DSB induction. Chromatin condensation in mut KRAS cells was characterized by a mitosis-like colocalization of H3K9me3 and p-H3S10 outside of the G2/M phase of the cell cycle. The dependence of H3K9me3/p-H3S10 on EGFR was confirmed in KRAS mut xenografts. Consistent with its expression in G1 Aurora B promoted the co-localization signal in a manner that was codependent on EGFR and PKCa. PKCa in addition to MEK/ERK was required for suppression of DSB-triggered premature senescence by EGFR. Blockade of autophagy resulted in a KRAS mut dependent senescence-to-apoptosis switch in cells treated with IR/erlotinib, increasing the IR enhancement factor to 1.54-2.00 (p < 0.05). Conclusions: We have identified an interphase chromatin condensation pathway that includes EGFR, Aurora B, and PKCa as a cause of mut KRAS dependent IR resistance. These findings may reposition EGFR inhibitors for combination with DSB-inducing therapies in KRAS mut cancer and yield novel therapeutic opportunities. Acknowledgment: This research was supported by DF/HCC SPORE in Lung Cancer, P50 CA090578, American Cancer Society 123420RSG-12224-01-DMC, UK Wellcome Trust 086357, Deutsche Forschungsgemeinschaft DFG-PAK-190, Federal Share of program income earned by MGH on C06 CA059267, Proton Therapy Research & Treatment Center. Author Disclosure: H. Willers: None. M. Wang: None. J. Han: None. A. Kern: None. M. Hu¨lsko¨tter: None. M. Krause: None. M. Baumann: None. C. Benes: None. J. Efstathiou: None. J. Settleman: A. Employee; Genentech.
223 Chromatin Condensation Mediated by EGFR and Aurora B Protects KRAS-Mutant Lung Cancer Cells Against Ionizing Radiation H. Willers,1 M. Wang,1 J. Han,1 A. Kern,1 M. Hu¨lsko¨tter,1 M. Krause,2 M. Baumann,2 C. Benes,3 J. Efstathiou,1 and J. Settleman3; 1 Massachusetts General Hospital, Boston, MA, 2Technical University Dresden, Dresden, Germany, 3Massachusetts General Hospital Cancer Center, Charlestown, MA Purpose/Objective(s): Combinations of EGFR targeted drugs and ionizing radiation (IR) have produced inconsistent results in patients. Genomic biomarkers and novel approaches to augment radiosensitization are needed. Materials/Methods: We screened 37 annotated lung cancer cell lines. We also utilized 3 isogenic models of mutated (mut) vs wild-type (wt) KRAS (using protein overexpression, depletion, and allelic deletion). KRAS mut A549 cells were grown as xenografts. Pharmacologic inhibitors of EGFR, Aurora B, PKCa, MEK were used. We assayed radiosensitization using clonogenic survival and short-term endpoints. DNA double-strand breaks (DSB) were monitored by employing gH2AX and 53BP1 foci and the Comet assay. Chromatin state was characterized by transmission electron and immunofluorescence microscopy and Western blotting. We utilized flow cytometry for studying cell cycle profiles and apoptosis and b-galactosidase staining for detection of senescent cells. Results: EGFR targeted drugs erlotinib and cetuximab enhanced the effects of IR in KRAS mut (n Z 13) but not wt (n Z 24) cell lines, with enhancement factors of 1.08 and 1.12 vs 0.96 and 1.00, respectively (p < 0.01). KRAS mut specific radiosensitization was confirmed in isogenic models including tumor spheres. Surprisingly, EGFR inhibition caused a w1.4-fold increase in DSB induction in KRAS mut but not wt cells, which was independent of non-homologous end-joining and EGFR nuclear translocation. Increased DSB induction correlated with a reduction in condensed chromatin prior to IR. Pharmacologic chromatin relaxation
224 NRF2 Is a Novel Oncogene and Biomarker of Therapeutic Resistance in Non-Small Cell Lung Cancer M. Abazeed,1 C. Xu,2 D. Adams,3 P. Tamayo,3 J. Loeffler,4 J. Suh,1 M. Meyerson,2 K. Wong,2 and P. Hammerman2; 1Cleveland Clinic Foundation, Cleveland, OH, 2Dana-Farber Cancer Institute, Boston, MA, 3 Broad Institute, Cambridge, MA, 4Massachusetts General Hospital, Boston, MA Purpose/Objectives(s): Radiation therapy is one of the mainstays of anticancer treatment, but the relationship between the radiosensitivity of cancer cells and their genomic characteristics is not well defined. To accelerate discovery of genotype-directed radiation therapy, or precision radiotherapeutics, systematic approaches are needed to identify genetic features that confer radiotherapeutic resistance and target these features with small-molecule drugs. Materials/Methods: We developed a systematic high-throughput method for profiling radiation sensitivity and benchmarked this method against the conventional clonogenic survival assay. We combined results from this high-throughput assay with genomic parameters in cell lines from squamous cell lung carcinoma, which is standardly treated by radiation therapy, to identify parameters that predict radiation sensitivity. Results: We identified the frequent presence (32%) of mutations in NFE2L2 (encoding the antioxidant- and detoxification-inducing transcription factor, NRF2) and KEAP1, a NRF2 binding partner, in squamous cell lung cancer. These data were generated using whole-exome and directed sequencing of 176 squamous cell lung cancers (The Cancer Genome Atlas [TCGA]) and 20 lung squamous cancer cell lines, respectively. We showed that gain-of-function mutations in NFE2L2 make lung cancer cells confer resistance to radiation. NFE2L2 knockdown resulted in growth arrest and radiation sensitivity in cell lines with NFE2L2 mutation but not in wild-type cell lines. An expression-based, in silico screen nominated inhibitors of PI3K as NFE2L2 antagonists. We showed that the selective PI3K inhibitor, NVP-BKM120, both decreased NRF2 protein levels and sensitized NFE2L2 or KEAP1 mutant cells to radiation in a GSK-3b dependent manner. We developed transgenic mice expressing a gain-of-function NFE2L2 alleles. We bred the NFE2L2 transgenic with a strain expressing a conditional allele of TP53 and found that the mice developed lung carcinomas, implicating NFE2L2, for the first time, in lung oncogenesis. Analysis of TCGA data indicates that
Volume 90 Number 1S Supplement 2014 alterations in NFE2L2 and KEAP1 confer poorer overall survival (P Z 0.02). Conclusions: The integrative, high-throughput methods shown here for large-scale profiling of radiation survival and genomic features of solidtumor derived cell lines should facilitate tumor radiogenomics and the discovery of radiation sensitizers and protective agents. The potential clinical translation of these findings includes the development of NFE2L2 molecular diagnostics and the prospective validation of the first radiotherapeutic resistance biomarker in lung NSCLC. Author Disclosure: M. Abazeed: None. C. Xu: None. D. Adams: None. P. Tamayo: None. J. Loeffler: None. J. Suh: None. M. Meyerson: G. Consultant; MM is a consultant to Foundation Medicine. R. Ownership Other; MM is an equity holder in Foundation Medicine. K. Wong: None. P. Hammerman: G. Consultant; P.S.H. reports consulting fees from ARIAD.
225 Induction of HO-1 and CO Synthesis Radiosensitizes Tumor Epithelial Cells With CO Acting as a NO Mimetic R.B. Mikkelsen,1 C.S. Rabender,1 P. Graves,2 and M.S. Anscher1; 1 Virginia Commonwealth University, Richmond, VA, 2New York Methodist Hospital, Brooklyn, NY Purpose/Objective(s): A previous proteomic study on radiation-induced lung injury in rats demonstrated that radiation stimulated a robust but transient expression of heme oxygenase-1 (HO-1). Hemin also induces HO-1 expression and is being evaluated clinically in the treatment of porphyria, a number of inflammatory diseases and ischemia-reperfusion. Since HO-1 also has anti-fibrotic properties, its induction may have a role in preventing radiation-induced lung injury. As a first step this study tests whether induction of HO-1 sensitizes or protects lung carcinoma cells from radiation. Materials/Methods: Cell survival of A549 lung carcinoma cells after a single radiation dose (5 Gy) was examined by traditional clonogenic assay with and without a 24 h pre-radiation treatment with 10 uM hemin. HO-1 expression was monitored by Western blot, reactive oxygen generation by HPLC/fluorescence assay of superoxide oxidation products of dihydroethidium and cGMP by ELISA as a test for activation of NO-dependent soluble guanylate cyclase (sGC). The thorax of C57Bl/j6 mice was irradiated using in 4 fractions of 7 Gy. Mice were treated after radiation with or without hemin given ip (25 mg/kg) and at 10 weeks post-irradiation mice were sacrificed, lungs embedded in OCT and evaluated immunologically for TGF-b expression. Results: Hemin induces a robust HO-1 expression in A549 cells with maximal expression at 24 hrs. Clonogenic survival after a single 5 Gy exposure is 27.8 1.2 (n Z 6) and this is not changed by simultaneous treatment with hemin. If cells are pre-treated with hemin 24 hrs prior to radiation or 24 hrs after radiation, cell survival is reduced respectively to 14.9 0.7 (n Z 6, p < .001) and 17.5 1.2 (n Z 6, p < .002). Superoxide expression in cells treated with hemin for 24 hrs is reduced by more than 50% as determined by HPLC analysis dihydoethidum oxidation products. cGMP levels more than doubled from 1.26 pmol/mg protein to 4.65 pmol/mg protein (n Z 4, p < 0.001) by a mechanism inhibited by the sGC inhibitor 1H- [1, 2, 4]oxadiazolo[4, 3- a]quinoxalin-1-one. Animal studies demonstrate that inducing HO-1 expression blocks radiation-induced TGFb expression at 10 weeks post-irradiation. Conclusions: Induction of HO-1 enhances the cytotoxic effect of radiation on lung tumor A549 cells while at the same time reducing expression of a pro-fibrotic molecule important in radiation induced lung injury. We speculate that HO-1 generation of CO by binding to the heme of NOS inhibits the pro-inflammatory activities of this enzyme as indicated by reduced superoxide, while at the same time CO acting as a NO mimetic binds to the heme of sGC activates the anti-inflammatory cGMP/protein kinase G pathway inhibiting TGFb signaling. These results suggest a potential approach to treating radiation-induced lung injury.
Oral Scientific Sessions S105 Author Disclosure: R.B. Mikkelsen: None. C.S. Rabender: Graves: None. M.S. Anscher: K. Advisory Board; Scientific Board and Stock Options for CivaTech Oncology Scientific Board of Virginia Life Sciences Investments. N. Stock Options; Advisory Board and Stock Options for CivaTech Oncology Advisory Board of Virginia Life Sciences Investments.
None. P. Advisory Advisory Scientific Scientific
226 Serum VEGF Associated With Survival in Non-Small Cell Lung Cancer Patients Treated With Definitive Chemoradiation Therapy T. Xu, D. Gomez, M. O’Reilly, Q. Nguyen, L.B. Levy, R. Komaki, R. Mohan, and Z. Liao; University of Texas MD Anderson Cancer Center, Houston, TX Purpose/Objective(s): We have previously shown that both TGF-b1 and VEGF are associated with tumor burden and response on positron emission tomography (PET) after chemoradiation therapy (CRT) in non-small-cell lung cancer (NSCLC). We sought to determine if changes in these factors during CRT are correlated with survival. Materials/Methods: We included fifty five patients treated with definitive CRT (radiation dose 60 Gy) on a prospective clinical trial. Serial blood samples were collected pre-CRT, 2-3 times during, and 1-3 months postCRT. Cytokines were measured in duplicates using a solid phase-based multiplex platform. The mean concentrations of cytokines during CRT were used as the “during” treatment level. Cox regression was performed to evaluate the association between cytokine levels and both progression free survival (PFS) and overall survival (OS) while adjusting for the following factors: age, gender, race, tumor size, histology, stage, smoking pack year, receiving of induction chemotherapy, radiation technique and total delivered dose. Results: The median follow-up time was 27 (range, 15-38) months, the median overall survival (OS) time was 28 months, and the median progression free survival (PFS) time was 10.4 months. We found that VEGF baseline and mean levels during CRT were associated with both OS (baseline: HR Z 2.17, 95% CI Z 1.22-3.89, P Z 0.008; during: HR Z 4.05, 95% CI Z 1.52-10.78, P Z 0.005) and PFS (baseline: HR Z 1.68, 95% CI Z 1.05-2.70, P Z 0.03; during: HR Z 2.56, 95% CI Z 1.21-5.42, P Z 0.014) after adjustment of clinical covariates. In addition, the percentage decrease of VEGF from pre to post-CRT was also associated with OS (HR Z 0.24, 95% CI Z 0.08-0.70, P Z 0.009). VEGF post-CRT was not associated with OS or PFS, nor was TGF-b1 levels at any time point (p > 0.05). Conclusions: In patients with locally advanced NSCLC receiving definitive chemoradiation, absolute VEGF levels before and during RT, and the percentage change of VEGF during treatment, were associated with both PFS and OS. Similar trends were not observed with TGF-b1. Author Disclosure: T. Xu: None. D. Gomez: None. M. O’Reilly: None. Q. Nguyen: None. L.B. Levy: None. R. Komaki: None. R. Mohan: None. Z. Liao: None.
227 Automatic Determination of Beam Angles in Lung IMRT Planning L. Yuan,1 Y. Ge,2 F. Yin,1 Y. Li,3 Y. Sheng,4 C.R. Kelsey,1 and Q.J. Wu1; 1 Duke University Medical Center, Durham, NC, 2University of North Carolina at Charlotte, Charlotte, NC, 3The First Affiliated Hospital of Chongqing Medical University, Chongqing, China, 4Duke University Medical Physics Program, Durham, NC Purpose/Objective(s): To develop a technique to automatically determine beam angle configurations customized for patient-specific anatomy and tumor geometry in lung IMRT planning. Materials/Methods: The relationship between individual patient anatomy and optimal beam configurations are learned from a set of high quality clinical plans. It involves three major steps: Step 1: Establish a beam configuration atlas using 60 clinical lung IMRT plans consisting of cases with tumor locations in the right lung, left lung, mediastinum and chest wall (26, 23, 8, and 3 cases, respectively). The volume of the PTV ranges
International Journal of Radiation Oncology Biology Physics
embryo fibroblasts (MEFs). The stress response to ionizing radiation YTR107 was assessed in wild-type and NPM-null MEFs and 6 NSCLC cell lines by quantifying pNPM1, gamma-H2AX, and Rad51 foci, neutral comet tail moment, and colony formation. In vivo efficacy of YTR107 radiosensitization was tested in a syngeneic xenograft model of tumor growth delay. NPM1 levels in a human-derived NSCLC tissue microarray (TMA) were determined by immunohistochemistry. Results: We observed that proliferating NPM1-null MEFs, but not wildtype MEFs, have high levels of gamma-H2AX foci, a characteristic of replication stress. In addition, NPM1-null MEFs exhibit increased radiation sensitivity via a defect in Rad51-mediated repair of DNA DSBs. We further determined that YTR107-mediated radiosensitization requires NPM1. The DNA damage response pathway is continuously activated in NSCLC due to constant replication stress. Because NPM1 expression minimizes replication stress, we further hypothesized that NSCLC tumors may have significant expression of NPM1. This was validated by analyzing a NSCLC TMA that demonstrated very low NPM1 expression in matched normal lung parenchyma but significantly increased NPM1 expression in Stage III NSCLC cores. We then observed that YTR107-mediated targeting of NPM1 resulted in radiosensitization in 6 NSCLC cell lines in vitro and in a syngeneic tumor model in vivo. Conclusions: These data demonstrate genetic evidence of NPM1 contribution to repair of radiation-induced DNA damage and suggest that NPM1 represents a potential therapeutic target for radiosensitization of NSCLC. Author Disclosure: C.A. Smith: None. K.R. Sekhar: Q. Patent/License Fee/Copyright; Co-inventor on the patent covering YTR107. M. Benamar: None. A. Venkateswaran: None. S. Sasi: None. N.R. Penthala: Q. Patent/License Fee/Copyright; Co-inventor on the patent covering YTR107. P.A. Crooks: Q. Patent/License Fee/Copyright; Co-inventor on the patent covering YTR107. S.R. Hann: None. L. Geng: None. T. Abbas: None. M.L. Freeman: Q. Patent/License Fee/Copyright; Co-inventor on the patent covering YTR107.
abrogated the effect of EGFR inhibition on DSB induction. Chromatin condensation in mut KRAS cells was characterized by a mitosis-like colocalization of H3K9me3 and p-H3S10 outside of the G2/M phase of the cell cycle. The dependence of H3K9me3/p-H3S10 on EGFR was confirmed in KRAS mut xenografts. Consistent with its expression in G1 Aurora B promoted the co-localization signal in a manner that was codependent on EGFR and PKCa. PKCa in addition to MEK/ERK was required for suppression of DSB-triggered premature senescence by EGFR. Blockade of autophagy resulted in a KRAS mut dependent senescence-to-apoptosis switch in cells treated with IR/erlotinib, increasing the IR enhancement factor to 1.54-2.00 (p < 0.05). Conclusions: We have identified an interphase chromatin condensation pathway that includes EGFR, Aurora B, and PKCa as a cause of mut KRAS dependent IR resistance. These findings may reposition EGFR inhibitors for combination with DSB-inducing therapies in KRAS mut cancer and yield novel therapeutic opportunities. Acknowledgment: This research was supported by DF/HCC SPORE in Lung Cancer, P50 CA090578, American Cancer Society 123420RSG-12224-01-DMC, UK Wellcome Trust 086357, Deutsche Forschungsgemeinschaft DFG-PAK-190, Federal Share of program income earned by MGH on C06 CA059267, Proton Therapy Research & Treatment Center. Author Disclosure: H. Willers: None. M. Wang: None. J. Han: None. A. Kern: None. M. Hu¨lsko¨tter: None. M. Krause: None. M. Baumann: None. C. Benes: None. J. Efstathiou: None. J. Settleman: A. Employee; Genentech.
223 Chromatin Condensation Mediated by EGFR and Aurora B Protects KRAS-Mutant Lung Cancer Cells Against Ionizing Radiation H. Willers,1 M. Wang,1 J. Han,1 A. Kern,1 M. Hu¨lsko¨tter,1 M. Krause,2 M. Baumann,2 C. Benes,3 J. Efstathiou,1 and J. Settleman3; 1 Massachusetts General Hospital, Boston, MA, 2Technical University Dresden, Dresden, Germany, 3Massachusetts General Hospital Cancer Center, Charlestown, MA Purpose/Objective(s): Combinations of EGFR targeted drugs and ionizing radiation (IR) have produced inconsistent results in patients. Genomic biomarkers and novel approaches to augment radiosensitization are needed. Materials/Methods: We screened 37 annotated lung cancer cell lines. We also utilized 3 isogenic models of mutated (mut) vs wild-type (wt) KRAS (using protein overexpression, depletion, and allelic deletion). KRAS mut A549 cells were grown as xenografts. Pharmacologic inhibitors of EGFR, Aurora B, PKCa, MEK were used. We assayed radiosensitization using clonogenic survival and short-term endpoints. DNA double-strand breaks (DSB) were monitored by employing gH2AX and 53BP1 foci and the Comet assay. Chromatin state was characterized by transmission electron and immunofluorescence microscopy and Western blotting. We utilized flow cytometry for studying cell cycle profiles and apoptosis and b-galactosidase staining for detection of senescent cells. Results: EGFR targeted drugs erlotinib and cetuximab enhanced the effects of IR in KRAS mut (n Z 13) but not wt (n Z 24) cell lines, with enhancement factors of 1.08 and 1.12 vs 0.96 and 1.00, respectively (p < 0.01). KRAS mut specific radiosensitization was confirmed in isogenic models including tumor spheres. Surprisingly, EGFR inhibition caused a w1.4-fold increase in DSB induction in KRAS mut but not wt cells, which was independent of non-homologous end-joining and EGFR nuclear translocation. Increased DSB induction correlated with a reduction in condensed chromatin prior to IR. Pharmacologic chromatin relaxation
224 NRF2 Is a Novel Oncogene and Biomarker of Therapeutic Resistance in Non-Small Cell Lung Cancer M. Abazeed,1 C. Xu,2 D. Adams,3 P. Tamayo,3 J. Loeffler,4 J. Suh,1 M. Meyerson,2 K. Wong,2 and P. Hammerman2; 1Cleveland Clinic Foundation, Cleveland, OH, 2Dana-Farber Cancer Institute, Boston, MA, 3 Broad Institute, Cambridge, MA, 4Massachusetts General Hospital, Boston, MA Purpose/Objectives(s): Radiation therapy is one of the mainstays of anticancer treatment, but the relationship between the radiosensitivity of cancer cells and their genomic characteristics is not well defined. To accelerate discovery of genotype-directed radiation therapy, or precision radiotherapeutics, systematic approaches are needed to identify genetic features that confer radiotherapeutic resistance and target these features with small-molecule drugs. Materials/Methods: We developed a systematic high-throughput method for profiling radiation sensitivity and benchmarked this method against the conventional clonogenic survival assay. We combined results from this high-throughput assay with genomic parameters in cell lines from squamous cell lung carcinoma, which is standardly treated by radiation therapy, to identify parameters that predict radiation sensitivity. Results: We identified the frequent presence (32%) of mutations in NFE2L2 (encoding the antioxidant- and detoxification-inducing transcription factor, NRF2) and KEAP1, a NRF2 binding partner, in squamous cell lung cancer. These data were generated using whole-exome and directed sequencing of 176 squamous cell lung cancers (The Cancer Genome Atlas [TCGA]) and 20 lung squamous cancer cell lines, respectively. We showed that gain-of-function mutations in NFE2L2 make lung cancer cells confer resistance to radiation. NFE2L2 knockdown resulted in growth arrest and radiation sensitivity in cell lines with NFE2L2 mutation but not in wild-type cell lines. An expression-based, in silico screen nominated inhibitors of PI3K as NFE2L2 antagonists. We showed that the selective PI3K inhibitor, NVP-BKM120, both decreased NRF2 protein levels and sensitized NFE2L2 or KEAP1 mutant cells to radiation in a GSK-3b dependent manner. We developed transgenic mice expressing a gain-of-function NFE2L2 alleles. We bred the NFE2L2 transgenic with a strain expressing a conditional allele of TP53 and found that the mice developed lung carcinomas, implicating NFE2L2, for the first time, in lung oncogenesis. Analysis of TCGA data indicates that
Volume 90 Number 1S Supplement 2014 alterations in NFE2L2 and KEAP1 confer poorer overall survival (P Z 0.02). Conclusions: The integrative, high-throughput methods shown here for large-scale profiling of radiation survival and genomic features of solidtumor derived cell lines should facilitate tumor radiogenomics and the discovery of radiation sensitizers and protective agents. The potential clinical translation of these findings includes the development of NFE2L2 molecular diagnostics and the prospective validation of the first radiotherapeutic resistance biomarker in lung NSCLC. Author Disclosure: M. Abazeed: None. C. Xu: None. D. Adams: None. P. Tamayo: None. J. Loeffler: None. J. Suh: None. M. Meyerson: G. Consultant; MM is a consultant to Foundation Medicine. R. Ownership Other; MM is an equity holder in Foundation Medicine. K. Wong: None. P. Hammerman: G. Consultant; P.S.H. reports consulting fees from ARIAD.
225 Induction of HO-1 and CO Synthesis Radiosensitizes Tumor Epithelial Cells With CO Acting as a NO Mimetic R.B. Mikkelsen,1 C.S. Rabender,1 P. Graves,2 and M.S. Anscher1; 1 Virginia Commonwealth University, Richmond, VA, 2New York Methodist Hospital, Brooklyn, NY Purpose/Objective(s): A previous proteomic study on radiation-induced lung injury in rats demonstrated that radiation stimulated a robust but transient expression of heme oxygenase-1 (HO-1). Hemin also induces HO-1 expression and is being evaluated clinically in the treatment of porphyria, a number of inflammatory diseases and ischemia-reperfusion. Since HO-1 also has anti-fibrotic properties, its induction may have a role in preventing radiation-induced lung injury. As a first step this study tests whether induction of HO-1 sensitizes or protects lung carcinoma cells from radiation. Materials/Methods: Cell survival of A549 lung carcinoma cells after a single radiation dose (5 Gy) was examined by traditional clonogenic assay with and without a 24 h pre-radiation treatment with 10 uM hemin. HO-1 expression was monitored by Western blot, reactive oxygen generation by HPLC/fluorescence assay of superoxide oxidation products of dihydroethidium and cGMP by ELISA as a test for activation of NO-dependent soluble guanylate cyclase (sGC). The thorax of C57Bl/j6 mice was irradiated using in 4 fractions of 7 Gy. Mice were treated after radiation with or without hemin given ip (25 mg/kg) and at 10 weeks post-irradiation mice were sacrificed, lungs embedded in OCT and evaluated immunologically for TGF-b expression. Results: Hemin induces a robust HO-1 expression in A549 cells with maximal expression at 24 hrs. Clonogenic survival after a single 5 Gy exposure is 27.8 1.2 (n Z 6) and this is not changed by simultaneous treatment with hemin. If cells are pre-treated with hemin 24 hrs prior to radiation or 24 hrs after radiation, cell survival is reduced respectively to 14.9 0.7 (n Z 6, p < .001) and 17.5 1.2 (n Z 6, p < .002). Superoxide expression in cells treated with hemin for 24 hrs is reduced by more than 50% as determined by HPLC analysis dihydoethidum oxidation products. cGMP levels more than doubled from 1.26 pmol/mg protein to 4.65 pmol/mg protein (n Z 4, p < 0.001) by a mechanism inhibited by the sGC inhibitor 1H- [1, 2, 4]oxadiazolo[4, 3- a]quinoxalin-1-one. Animal studies demonstrate that inducing HO-1 expression blocks radiation-induced TGFb expression at 10 weeks post-irradiation. Conclusions: Induction of HO-1 enhances the cytotoxic effect of radiation on lung tumor A549 cells while at the same time reducing expression of a pro-fibrotic molecule important in radiation induced lung injury. We speculate that HO-1 generation of CO by binding to the heme of NOS inhibits the pro-inflammatory activities of this enzyme as indicated by reduced superoxide, while at the same time CO acting as a NO mimetic binds to the heme of sGC activates the anti-inflammatory cGMP/protein kinase G pathway inhibiting TGFb signaling. These results suggest a potential approach to treating radiation-induced lung injury.
Oral Scientific Sessions S105 Author Disclosure: R.B. Mikkelsen: None. C.S. Rabender: Graves: None. M.S. Anscher: K. Advisory Board; Scientific Board and Stock Options for CivaTech Oncology Scientific Board of Virginia Life Sciences Investments. N. Stock Options; Advisory Board and Stock Options for CivaTech Oncology Advisory Board of Virginia Life Sciences Investments.
None. P. Advisory Advisory Scientific Scientific
226 Serum VEGF Associated With Survival in Non-Small Cell Lung Cancer Patients Treated With Definitive Chemoradiation Therapy T. Xu, D. Gomez, M. O’Reilly, Q. Nguyen, L.B. Levy, R. Komaki, R. Mohan, and Z. Liao; University of Texas MD Anderson Cancer Center, Houston, TX Purpose/Objective(s): We have previously shown that both TGF-b1 and VEGF are associated with tumor burden and response on positron emission tomography (PET) after chemoradiation therapy (CRT) in non-small-cell lung cancer (NSCLC). We sought to determine if changes in these factors during CRT are correlated with survival. Materials/Methods: We included fifty five patients treated with definitive CRT (radiation dose 60 Gy) on a prospective clinical trial. Serial blood samples were collected pre-CRT, 2-3 times during, and 1-3 months postCRT. Cytokines were measured in duplicates using a solid phase-based multiplex platform. The mean concentrations of cytokines during CRT were used as the “during” treatment level. Cox regression was performed to evaluate the association between cytokine levels and both progression free survival (PFS) and overall survival (OS) while adjusting for the following factors: age, gender, race, tumor size, histology, stage, smoking pack year, receiving of induction chemotherapy, radiation technique and total delivered dose. Results: The median follow-up time was 27 (range, 15-38) months, the median overall survival (OS) time was 28 months, and the median progression free survival (PFS) time was 10.4 months. We found that VEGF baseline and mean levels during CRT were associated with both OS (baseline: HR Z 2.17, 95% CI Z 1.22-3.89, P Z 0.008; during: HR Z 4.05, 95% CI Z 1.52-10.78, P Z 0.005) and PFS (baseline: HR Z 1.68, 95% CI Z 1.05-2.70, P Z 0.03; during: HR Z 2.56, 95% CI Z 1.21-5.42, P Z 0.014) after adjustment of clinical covariates. In addition, the percentage decrease of VEGF from pre to post-CRT was also associated with OS (HR Z 0.24, 95% CI Z 0.08-0.70, P Z 0.009). VEGF post-CRT was not associated with OS or PFS, nor was TGF-b1 levels at any time point (p > 0.05). Conclusions: In patients with locally advanced NSCLC receiving definitive chemoradiation, absolute VEGF levels before and during RT, and the percentage change of VEGF during treatment, were associated with both PFS and OS. Similar trends were not observed with TGF-b1. Author Disclosure: T. Xu: None. D. Gomez: None. M. O’Reilly: None. Q. Nguyen: None. L.B. Levy: None. R. Komaki: None. R. Mohan: None. Z. Liao: None.
227 Automatic Determination of Beam Angles in Lung IMRT Planning L. Yuan,1 Y. Ge,2 F. Yin,1 Y. Li,3 Y. Sheng,4 C.R. Kelsey,1 and Q.J. Wu1; 1 Duke University Medical Center, Durham, NC, 2University of North Carolina at Charlotte, Charlotte, NC, 3The First Affiliated Hospital of Chongqing Medical University, Chongqing, China, 4Duke University Medical Physics Program, Durham, NC Purpose/Objective(s): To develop a technique to automatically determine beam angle configurations customized for patient-specific anatomy and tumor geometry in lung IMRT planning. Materials/Methods: The relationship between individual patient anatomy and optimal beam configurations are learned from a set of high quality clinical plans. It involves three major steps: Step 1: Establish a beam configuration atlas using 60 clinical lung IMRT plans consisting of cases with tumor locations in the right lung, left lung, mediastinum and chest wall (26, 23, 8, and 3 cases, respectively). The volume of the PTV ranges