- Email: [email protected]
Targeted Silencing of Artemis Enhanced Radiosensitivity in Human Colorectal Cancer Cells
E518
International Journal of Radiation Oncology Biology Physics
the clues that miR-203 could be involved in the post-transcriptional control of DNA repair pathway, PI3K-AKT pathway, SRC, and JAK/STAT3 pathway. Western analysis validated that miR-203 down regulated the expression of ATM, RAD51, PI3K, AKT, SRC, and STAT3. Overexpression of miR-203 inhibited of invasion and migration potential of U251cells and led to down-regulation of SLUG and Vimentin, and upregulation of Caludin-1 and ZO1. Conclusion: These data show miR-203 potentially controls DNA damage repair pathway, PI3K/AKT and JAK/STAT3 pathway, and those may collectively contribute to modulation of radiosensitivity in malignant glioma cells via inhibition of DNA damage repair, prosurvival signaling and EMT (Epithelial-Mesenchymal Transition) feature. Taken together, our findings suggest that miR-203 could be a useful target for overcoming the radioresistance of GBM. Work supported by grant (#2012-0004867 & #2013R1A1A2074531) from National Research Foundation, Korean Ministry of Future Creative Science to Kim IA. Author Disclosure: I. Kim: None. Y. Hwang: None. J. Jang: None. D. Kim: None. J. Park: None. D. Lee: None.
upon TP53 alteration (q < 1x10-12); NHEJ genes RAD50 and TP53BP1 were found to be significantly decreased upon TP53 alteration (q < 1x1014 ); NER genes DDB2, XPA and XPC were found to be significantly decreased upon TP53 alteration (q < 1x10-27); BER gene NEIL1 was found to be significantly decreased upon TP53 alteration (q < 1x10-20). When molecular subtype is considered, there are no significant changes in any these genes upon TP53 alteration for Basal-like, Her2-enriched, or Normal-like breast cancers. Upon TP53 alteration, NER genes DDB2, XPA, and XPC are significantly decreased in Luminal A (q < 0.01); NER genes DDB2 and XPC are significantly decreased in Luminal B (q < 0.05); BER gene NEIL1 is significantly decreased in Luminal A and Luminal B (q < 0.05). Conclusion: TP53 mutated breast tumors have elevated levels of HR components and decreased levels of NHEJ, BER, and NER components. Luminal A and B subtypes have decreased levels of NER and BER components when TP53 is mutated. Author Disclosure: N.T. Pfister: None. K. Regunath: None. E.P. Connolly: None. C. Prives: None.
3298
3297 TP53 Status Predicts DNA Damage Response Pathways in Breast Cancer N.T. Pfister,1,2 K. Regunath,2 E.P. Connolly,3 and C. Prives2; 1Columbia University Medical Center, New York, NY, 2Columbia University, New York, NY, 3NewYork-Presbyterian Hospital Columbia Campus, New York, NY Purpose/Objective(s): p53 is the primary mediator of cellular response to radiation insult. Therefore we investigated whether TP53 mutational status impacts the expression of DNA damage pathway components in breast tumors by using The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma Provisional Database. Materials/Methods: TCGA somatic mutation and RNA-sequence V2 datasets were analyzed in MatLab. Stratification was performed based on tumor staging, UCSC PAM50 molecular subtype classification, and TP53 mutation status. TP53 mutations (missense, nonsense, frameshift, inframe deletion, and in-frame insertion) were combined into the TP53 Alteration group; TP53 Wild-Type status was assumed if there were no detected mutations. RNA expression values were analyzed as upper quartile normalized RNA-Seq by Expectation-Maximization (RSEM). The median gene expression was calculated for each gene of interest. The two-tailed t-test was utilized and the p-value was adjusted for multiple testing by using the Benjamini-Hochberg false discovery rate procedure (q-value). Canonical DNA damage response genes were categorized as nucleotide excision repair (NER), base excision repair (BER), homologous recombination (HR), or non-homologous end joining (NHEJ). Results: The Table reports the number of samples by stage and molecular subtype classification. As expected, the Basal-like subtype has an increased frequency of TP53 mutations, and TP53 mutation frequency is similar among tumor stages. Comparing TP53 Wild-Type vs. Altered (all stages combined): Expression of homologous recombination pathway genes RAD51, EXO1, and BLM were found to be significantly increased
Targeted Silencing of Artemis Enhanced Radiosensitivity in Human Colorectal Cancer Cells H. Liu, X. Sun, H. Gao, T. Jiang, B. Gu, Q. Yang, and S. Zheng; Sir Run Run Shaw Hospital, Hangzhou, China Purpose/Objective(s): Artemis-deficient human cells displayed hypersensitivity to many DNA damaging agents, like etoposide and bleomycin, and irradiation. However, how modification of Artemis affects radiosensitivity in cancer cells has not been largely investigated. This study aimed to investigate the expression of Artemis and the effect of silencing Artemis on cells radiosensitivity in human colorectal cancer. Materials/Methods: We assess the expression of Artemis in cancer and adjacent normal tissues by quantitative PCR in 50 cases of human colorectal carcinoma samples. Using lentivirus-mediated siRNA, we knockdown Artemis in human colorectal cancer cell line, RKO. Colony-forming assay, MTT assay and in vivo assay with nude mice were done to investigate sensitivity to IR and other DNA damaging agent in Artemisknockdown RKO cells. g-H2AX foci assay was done to study DSB rejoining. Through apoptosis detection assay and Western blot, we study the impact of silencing Artemis on apoptosis signaling pathways induced by DNA-damaging agent in RKO cells. Results: We found that expression of Artemis in 28% of colon cancer is higher in cancer tissue than adjacent normal tissues. The increased radiosensitivity to irradiation and other DNA damaging agents was found in Artemis-knockdown RKO cells in vitro and in vivo assay. We also found the delayed DSBs rejoining in Artemis-knockdown RKO cells by g-H2AX foci assay. Increased apoptosis happened in Artemisknockdown RKO cells and p53/p21 signaling pathway was involved in this process. Conclusion: In this study, we demonstrated that Artemis expression was higher in part of colorectal cancer tissue than adjacent normal tissue. Silencing Artemis can increase cell radiosensitivity in human colorectal cancer. Selective targeting of Artemis has the potential to sensitize tumors to radio/chemotherapy.
Poster Viewing Abstracts 3297; Table 1 TP53 Wild-Type TP53 Alteration TP53 Wild-Type TP53 Alteration
Stage I 119 41 Basal-like 30 103
Stage II 369 169 Her2-enriched 23 43
Stage III 147 62 Luminal A 366 42
Stage IV 8 7 Luminal B 126 59
Normal-like 17 6
Total 643 279 Total 562 253
Volume 93 Number 3S Supplement 2015 Author Disclosure: H. Liu: None. X. Sun: None. H. Gao: None. T. Jiang: None. B. Gu: None. Q. Yang: None. S. Zheng: None.
3299 Gene Expression Profiling Implicates the Tumor Suppressor miR-206 in HPV+ Oropharyngeal Squamous Cell Carcinoma Distant Recurrence S.S. Khwaja, H. Zhao, H.A. Gay, C.R. Spencer, W.L. Thorstad, J.S. Lewis, Jr, and X. Wang; Washington University School of Medicine, St. Louis, MO Purpose/Objective(s): Although HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients have an excellent prognosis, 10-15% of patients will develop distant recurrence despite standard of care therapy. Using high throughput gene expression profiling, we aim to identify novel molecular pathways involved in metastatic HPV+ OPSCC. Materials/Methods: Thirty-nine patients with stage III/IV HPV+ OPSCC treated definitively with chemoradiation (20%) or with post-operative radiation +/- chemo (80%) were included in this study. HPV status was determined by quantitative real-time PCR (qPCR) using RNA extracted from formalin-fixed paraffin-embedded (FFPE) specimens. Nineteen patients had biopsy-proven metastatic disease (DM+, 49%) and 20 with no recurrence (DM-, 51%). The 19 DM+-paired distant metastatic tumor tissues (M) were also included in this study. RNA extracted from DM+ and DM- primaries as well as M were prepared for gene expression studies using the established RNA-seq technique. Degree of differential gene expression in DM+/DM- and DM+/M was determined with Student’s t-test. Gene set enrichment analysis (GSEA) was performed on the differential expression data. Enriched gene sets were defined as having a p-value < 0.05 and a false discovery ratio (FDR) < 0.25. Normalized enrichment score (NES) indicated the degree of enrichment normalized for gene set size. Profiling of 384 micro RNAs (miRNAs) in DM+/M was accomplished using qPCR. Results: Median time to distant recurrence in DM+ was 1 yr (0.02-2.5 yrs), with the most common sites of metastasis being lung (63%), bone (16%), and liver (11%). Patients in DM- showed no evidence of disease after a median follow-up of 5 yrs. The M group consisted of metastatic tissue in lung (nZ14), bone (nZ3), mediastinal lymph nodes (nZ1), and thyroid (nZ1). Over 600 genes were differentially expressed between DM+/DMprimaries. The top 45 differentially expressed genes from RNA-seq, with cellular roles in vesicle trafficking, transcriptional regulation, RNA processing, stress response, cell proliferation, and chromatin remodeling, were validated with qPCR with a correlation of 0.77. GSEA studies of DM+/DM- revealed enrichment of the metastasis-associated miRNA gene sets miR-34B (NES 1.9, p<0.001, and FDRZ0.1) and miR-206 (NES 1.8, p Z 0.002, and FDR Z 0.1). Further supporting the role of miR-206 in metastasis, profiling of 384 miRNAs in DM+/M revealed miR-206 to be the most differentially-expressed with a 6-fold decrease in expression in M vs DM+ (p<0.001). Conclusion: We present evidence implicating the tumor suppressor miR206 in HPV+ OPSCC distant recurrence. Further investigation of this miRNA and its target genes are warranted to improve our understanding of mechanisms of distant metastasis in HPV+ OPSCC. Author Disclosure: S.S. Khwaja: None. H. Zhao: None. H.A. Gay: None. C.R. Spencer: None. W.L. Thorstad: None. J.S. Lewis Jr.: None. X. Wang: None.
Poster Viewing Session E519
3300 Unraveling the Mechanism of SBRT Response in EGFR-Addicted Pulmonary Adenocarcinoma by CRISPR/Cas9 Functional Genomics A.L. Polishchuk, J. Flanagan, L.A. Gilbert, M. Horlbeck, X. Yang, A. Olow, M. Bassik, J.S. Weissman, D.A. Haas-Kogan, J.A. Wells, and T.G. Bivona; University of California, San Francisco, San Francisco, CA Purpose/Objective(s): Stereotactic body radiation therapy (SBRT) has revolutionized the treatment of non-small cell lung cancer (NSCLC). EGFR driver mutations portend favorable treatment response after hypofractionated irradiation of NSCLC lesions. However, clinical outcomes of concurrent EGFR inhibition and radiation therapy in NSCLC have been variable leaving the role of EGFR addiction in mediating response to hypofractionated radiation unclear. To test the significance of EGFR signaling and systematically identify genetic loci that impact SBRT response in an EGFR mutant patient-derived NSCLC model, we have performed an unbiased radiation resistance screen using CRISPR/Cas9 genetic lesioning. Because of direct, DNA-level gene inactivation, the CRISPR/Cas9 system is expected to offer superior sensitivity over prior RNAi-based functional genomic techniques. Materials/Methods: Radiation sensitivities of the HCC827 (EGFR del exon 19) pulmonary adenocarcinoma cell line, and of a variant stably transduced with Cas9/control sgRNA were measured with clonogenic assays. HCC827 cells stably expressing Cas9 were transduced with an exome-targeting w80,000 sgRNA library (4 sgRNA/gene for w20,000 genes). A 10-day screen for differential survival after a single dose of 10 Gy was performed. Measurement of bar coded sgRNA abundance (to identify sgRNA depletion or enrichment) was made by next-generation deep sequencing of isolated genomic DNA from cell populations harvested during the screen. Results: Comparable surviving fractions of parent and Cas9/control sgRNA transduced variants were observed after irradiation to 10 Gy x 1, suggesting that Cas9 does not intrinsically perturb radiation sensitivity at high doses. As an internal validation of our screen, sgRNAs targeting the driver oncogene EGFR (required for survival in HCC827 cells) were depleted in the non-irradiated condition at day 10 compared with day 0 (pZ0.005). Interestingly, sgRNAs targeting EGFR were neither significantly enriched nor depleted in the irradiated vs. unirradiated comparison (pZ0.72). As further validation, the most significantly enriched sgRNA set in the irradiated versus non-irradiated cell populations targeted SERPINF1 (pZ0.002), a recently identified enhancer of radiosensitivity in NSCLC. A highly depleted sgRNA set in the same comparison targeted RB1 (pZ0.01), which has an essential role in DNA damage repair. Additional novel radio-efficacy targets were identified and are currently under investigation. Conclusion: Mutated EGFR does not appear to directly modulate SBRT response in an EGFR-addicted NSCLC model. Interrogation of downstream EGFR pathway elements and validation of putative novel molecular determinants of SBRT response identified from the screen is ongoing. Author Disclosure: A.L. Polishchuk: Employee; Peninsula Orthopedic Associates. Research Grant; Lung Cancer Research Foundation. J. Flanagan: None. L.A. Gilbert: None. M. Horlbeck: None. X. Yang: None. A. Olow: None. M. Bassik: None. J.S. Weissman: None. D.A. HaasKogan: Employee; UCSF. J.A. Wells: None. T.G. Bivona: Employee; Plexikkon. Research Grant; Addario Foundation, Kinship Foundation, HHMI.
International Journal of Radiation Oncology Biology Physics
the clues that miR-203 could be involved in the post-transcriptional control of DNA repair pathway, PI3K-AKT pathway, SRC, and JAK/STAT3 pathway. Western analysis validated that miR-203 down regulated the expression of ATM, RAD51, PI3K, AKT, SRC, and STAT3. Overexpression of miR-203 inhibited of invasion and migration potential of U251cells and led to down-regulation of SLUG and Vimentin, and upregulation of Caludin-1 and ZO1. Conclusion: These data show miR-203 potentially controls DNA damage repair pathway, PI3K/AKT and JAK/STAT3 pathway, and those may collectively contribute to modulation of radiosensitivity in malignant glioma cells via inhibition of DNA damage repair, prosurvival signaling and EMT (Epithelial-Mesenchymal Transition) feature. Taken together, our findings suggest that miR-203 could be a useful target for overcoming the radioresistance of GBM. Work supported by grant (#2012-0004867 & #2013R1A1A2074531) from National Research Foundation, Korean Ministry of Future Creative Science to Kim IA. Author Disclosure: I. Kim: None. Y. Hwang: None. J. Jang: None. D. Kim: None. J. Park: None. D. Lee: None.
upon TP53 alteration (q < 1x10-12); NHEJ genes RAD50 and TP53BP1 were found to be significantly decreased upon TP53 alteration (q < 1x1014 ); NER genes DDB2, XPA and XPC were found to be significantly decreased upon TP53 alteration (q < 1x10-27); BER gene NEIL1 was found to be significantly decreased upon TP53 alteration (q < 1x10-20). When molecular subtype is considered, there are no significant changes in any these genes upon TP53 alteration for Basal-like, Her2-enriched, or Normal-like breast cancers. Upon TP53 alteration, NER genes DDB2, XPA, and XPC are significantly decreased in Luminal A (q < 0.01); NER genes DDB2 and XPC are significantly decreased in Luminal B (q < 0.05); BER gene NEIL1 is significantly decreased in Luminal A and Luminal B (q < 0.05). Conclusion: TP53 mutated breast tumors have elevated levels of HR components and decreased levels of NHEJ, BER, and NER components. Luminal A and B subtypes have decreased levels of NER and BER components when TP53 is mutated. Author Disclosure: N.T. Pfister: None. K. Regunath: None. E.P. Connolly: None. C. Prives: None.
3298
3297 TP53 Status Predicts DNA Damage Response Pathways in Breast Cancer N.T. Pfister,1,2 K. Regunath,2 E.P. Connolly,3 and C. Prives2; 1Columbia University Medical Center, New York, NY, 2Columbia University, New York, NY, 3NewYork-Presbyterian Hospital Columbia Campus, New York, NY Purpose/Objective(s): p53 is the primary mediator of cellular response to radiation insult. Therefore we investigated whether TP53 mutational status impacts the expression of DNA damage pathway components in breast tumors by using The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma Provisional Database. Materials/Methods: TCGA somatic mutation and RNA-sequence V2 datasets were analyzed in MatLab. Stratification was performed based on tumor staging, UCSC PAM50 molecular subtype classification, and TP53 mutation status. TP53 mutations (missense, nonsense, frameshift, inframe deletion, and in-frame insertion) were combined into the TP53 Alteration group; TP53 Wild-Type status was assumed if there were no detected mutations. RNA expression values were analyzed as upper quartile normalized RNA-Seq by Expectation-Maximization (RSEM). The median gene expression was calculated for each gene of interest. The two-tailed t-test was utilized and the p-value was adjusted for multiple testing by using the Benjamini-Hochberg false discovery rate procedure (q-value). Canonical DNA damage response genes were categorized as nucleotide excision repair (NER), base excision repair (BER), homologous recombination (HR), or non-homologous end joining (NHEJ). Results: The Table reports the number of samples by stage and molecular subtype classification. As expected, the Basal-like subtype has an increased frequency of TP53 mutations, and TP53 mutation frequency is similar among tumor stages. Comparing TP53 Wild-Type vs. Altered (all stages combined): Expression of homologous recombination pathway genes RAD51, EXO1, and BLM were found to be significantly increased
Targeted Silencing of Artemis Enhanced Radiosensitivity in Human Colorectal Cancer Cells H. Liu, X. Sun, H. Gao, T. Jiang, B. Gu, Q. Yang, and S. Zheng; Sir Run Run Shaw Hospital, Hangzhou, China Purpose/Objective(s): Artemis-deficient human cells displayed hypersensitivity to many DNA damaging agents, like etoposide and bleomycin, and irradiation. However, how modification of Artemis affects radiosensitivity in cancer cells has not been largely investigated. This study aimed to investigate the expression of Artemis and the effect of silencing Artemis on cells radiosensitivity in human colorectal cancer. Materials/Methods: We assess the expression of Artemis in cancer and adjacent normal tissues by quantitative PCR in 50 cases of human colorectal carcinoma samples. Using lentivirus-mediated siRNA, we knockdown Artemis in human colorectal cancer cell line, RKO. Colony-forming assay, MTT assay and in vivo assay with nude mice were done to investigate sensitivity to IR and other DNA damaging agent in Artemisknockdown RKO cells. g-H2AX foci assay was done to study DSB rejoining. Through apoptosis detection assay and Western blot, we study the impact of silencing Artemis on apoptosis signaling pathways induced by DNA-damaging agent in RKO cells. Results: We found that expression of Artemis in 28% of colon cancer is higher in cancer tissue than adjacent normal tissues. The increased radiosensitivity to irradiation and other DNA damaging agents was found in Artemis-knockdown RKO cells in vitro and in vivo assay. We also found the delayed DSBs rejoining in Artemis-knockdown RKO cells by g-H2AX foci assay. Increased apoptosis happened in Artemisknockdown RKO cells and p53/p21 signaling pathway was involved in this process. Conclusion: In this study, we demonstrated that Artemis expression was higher in part of colorectal cancer tissue than adjacent normal tissue. Silencing Artemis can increase cell radiosensitivity in human colorectal cancer. Selective targeting of Artemis has the potential to sensitize tumors to radio/chemotherapy.
Poster Viewing Abstracts 3297; Table 1 TP53 Wild-Type TP53 Alteration TP53 Wild-Type TP53 Alteration
Stage I 119 41 Basal-like 30 103
Stage II 369 169 Her2-enriched 23 43
Stage III 147 62 Luminal A 366 42
Stage IV 8 7 Luminal B 126 59
Normal-like 17 6
Total 643 279 Total 562 253
Volume 93 Number 3S Supplement 2015 Author Disclosure: H. Liu: None. X. Sun: None. H. Gao: None. T. Jiang: None. B. Gu: None. Q. Yang: None. S. Zheng: None.
3299 Gene Expression Profiling Implicates the Tumor Suppressor miR-206 in HPV+ Oropharyngeal Squamous Cell Carcinoma Distant Recurrence S.S. Khwaja, H. Zhao, H.A. Gay, C.R. Spencer, W.L. Thorstad, J.S. Lewis, Jr, and X. Wang; Washington University School of Medicine, St. Louis, MO Purpose/Objective(s): Although HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients have an excellent prognosis, 10-15% of patients will develop distant recurrence despite standard of care therapy. Using high throughput gene expression profiling, we aim to identify novel molecular pathways involved in metastatic HPV+ OPSCC. Materials/Methods: Thirty-nine patients with stage III/IV HPV+ OPSCC treated definitively with chemoradiation (20%) or with post-operative radiation +/- chemo (80%) were included in this study. HPV status was determined by quantitative real-time PCR (qPCR) using RNA extracted from formalin-fixed paraffin-embedded (FFPE) specimens. Nineteen patients had biopsy-proven metastatic disease (DM+, 49%) and 20 with no recurrence (DM-, 51%). The 19 DM+-paired distant metastatic tumor tissues (M) were also included in this study. RNA extracted from DM+ and DM- primaries as well as M were prepared for gene expression studies using the established RNA-seq technique. Degree of differential gene expression in DM+/DM- and DM+/M was determined with Student’s t-test. Gene set enrichment analysis (GSEA) was performed on the differential expression data. Enriched gene sets were defined as having a p-value < 0.05 and a false discovery ratio (FDR) < 0.25. Normalized enrichment score (NES) indicated the degree of enrichment normalized for gene set size. Profiling of 384 micro RNAs (miRNAs) in DM+/M was accomplished using qPCR. Results: Median time to distant recurrence in DM+ was 1 yr (0.02-2.5 yrs), with the most common sites of metastasis being lung (63%), bone (16%), and liver (11%). Patients in DM- showed no evidence of disease after a median follow-up of 5 yrs. The M group consisted of metastatic tissue in lung (nZ14), bone (nZ3), mediastinal lymph nodes (nZ1), and thyroid (nZ1). Over 600 genes were differentially expressed between DM+/DMprimaries. The top 45 differentially expressed genes from RNA-seq, with cellular roles in vesicle trafficking, transcriptional regulation, RNA processing, stress response, cell proliferation, and chromatin remodeling, were validated with qPCR with a correlation of 0.77. GSEA studies of DM+/DM- revealed enrichment of the metastasis-associated miRNA gene sets miR-34B (NES 1.9, p<0.001, and FDRZ0.1) and miR-206 (NES 1.8, p Z 0.002, and FDR Z 0.1). Further supporting the role of miR-206 in metastasis, profiling of 384 miRNAs in DM+/M revealed miR-206 to be the most differentially-expressed with a 6-fold decrease in expression in M vs DM+ (p<0.001). Conclusion: We present evidence implicating the tumor suppressor miR206 in HPV+ OPSCC distant recurrence. Further investigation of this miRNA and its target genes are warranted to improve our understanding of mechanisms of distant metastasis in HPV+ OPSCC. Author Disclosure: S.S. Khwaja: None. H. Zhao: None. H.A. Gay: None. C.R. Spencer: None. W.L. Thorstad: None. J.S. Lewis Jr.: None. X. Wang: None.
Poster Viewing Session E519
3300 Unraveling the Mechanism of SBRT Response in EGFR-Addicted Pulmonary Adenocarcinoma by CRISPR/Cas9 Functional Genomics A.L. Polishchuk, J. Flanagan, L.A. Gilbert, M. Horlbeck, X. Yang, A. Olow, M. Bassik, J.S. Weissman, D.A. Haas-Kogan, J.A. Wells, and T.G. Bivona; University of California, San Francisco, San Francisco, CA Purpose/Objective(s): Stereotactic body radiation therapy (SBRT) has revolutionized the treatment of non-small cell lung cancer (NSCLC). EGFR driver mutations portend favorable treatment response after hypofractionated irradiation of NSCLC lesions. However, clinical outcomes of concurrent EGFR inhibition and radiation therapy in NSCLC have been variable leaving the role of EGFR addiction in mediating response to hypofractionated radiation unclear. To test the significance of EGFR signaling and systematically identify genetic loci that impact SBRT response in an EGFR mutant patient-derived NSCLC model, we have performed an unbiased radiation resistance screen using CRISPR/Cas9 genetic lesioning. Because of direct, DNA-level gene inactivation, the CRISPR/Cas9 system is expected to offer superior sensitivity over prior RNAi-based functional genomic techniques. Materials/Methods: Radiation sensitivities of the HCC827 (EGFR del exon 19) pulmonary adenocarcinoma cell line, and of a variant stably transduced with Cas9/control sgRNA were measured with clonogenic assays. HCC827 cells stably expressing Cas9 were transduced with an exome-targeting w80,000 sgRNA library (4 sgRNA/gene for w20,000 genes). A 10-day screen for differential survival after a single dose of 10 Gy was performed. Measurement of bar coded sgRNA abundance (to identify sgRNA depletion or enrichment) was made by next-generation deep sequencing of isolated genomic DNA from cell populations harvested during the screen. Results: Comparable surviving fractions of parent and Cas9/control sgRNA transduced variants were observed after irradiation to 10 Gy x 1, suggesting that Cas9 does not intrinsically perturb radiation sensitivity at high doses. As an internal validation of our screen, sgRNAs targeting the driver oncogene EGFR (required for survival in HCC827 cells) were depleted in the non-irradiated condition at day 10 compared with day 0 (pZ0.005). Interestingly, sgRNAs targeting EGFR were neither significantly enriched nor depleted in the irradiated vs. unirradiated comparison (pZ0.72). As further validation, the most significantly enriched sgRNA set in the irradiated versus non-irradiated cell populations targeted SERPINF1 (pZ0.002), a recently identified enhancer of radiosensitivity in NSCLC. A highly depleted sgRNA set in the same comparison targeted RB1 (pZ0.01), which has an essential role in DNA damage repair. Additional novel radio-efficacy targets were identified and are currently under investigation. Conclusion: Mutated EGFR does not appear to directly modulate SBRT response in an EGFR-addicted NSCLC model. Interrogation of downstream EGFR pathway elements and validation of putative novel molecular determinants of SBRT response identified from the screen is ongoing. Author Disclosure: A.L. Polishchuk: Employee; Peninsula Orthopedic Associates. Research Grant; Lung Cancer Research Foundation. J. Flanagan: None. L.A. Gilbert: None. M. Horlbeck: None. X. Yang: None. A. Olow: None. M. Bassik: None. J.S. Weissman: None. D.A. HaasKogan: Employee; UCSF. J.A. Wells: None. T.G. Bivona: Employee; Plexikkon. Research Grant; Addario Foundation, Kinship Foundation, HHMI.