- Email: [email protected]
Twist1 Lung Adenocarcinoma Model via a CD8+ T Cell-Dependent Mechanism
S30
International Journal of Radiation Oncology Biology Physics
thresholding the week 12 TV for ADC values below the week 0 mean ADC (theoretically representing the active cellular component of tumor at diagnosis). Results: The median ADC (average for TV) at 0, 6 and 12 weeks was 1.48x10-3, 1.54x10-3 and 1.65x10-3 mm2/s with a generally increasing trend for ADC. ADC values between weeks 0 and 6 were significantly different (P<0.01), while values between weeks 0 and 12 (P Z 0.06) and weeks 6 and 12 (P Z 0.13) were not significant. ADC distributions and TVs are shown below.
inhibition). For the EJ5-GFP NHEJ reporter, Isce1-Trex2 (exonuclease) was used to generate blunt-ended DSBs. Percentages of GFP+ cells were measured by flow cytometry. Propidium iodide staining was used to measure cell cycle distribution. Results: Compared to HPV- HNSCC, HPV+ cases demonstrated a 33% increase in the proportion of deletions with flanking microhomology (n Z 949 deletions, 28% HPV+ vs 21% HPV-, P Z 0.005). TCGA RNAseq data revealed mRNA expression of POLQ to be 2.2 fold higher in HPV+ vs. HPV- cases (95% CI 2.1-2.4, P<0.001). Among 33 histologies of TCGA solid tumors, HPV+ HNSCC demonstrated the highest expression of POLQ (2.20.18 fold increase from HPV- HNSCC, P<0.001); cervical cancers were second highest. Using reporter assays, compared to empty vector (EV), expression of HPV16 E7 resulted in a decrease in NHEJ (-34%8%, P Z 0.003), and increases in HR (+8933%, P Z 0.002) and altEJ (+49%20%, P<0.001). Expression of the HPV16 E7 C24G mutant demonstrated no change in NHEJ (+0.3%9%, P Z 0.98), HR (+1845%, P Z 0.39) or altEJ (+1526%, P Z 0.24). Differences in DSB pathway utilization were not related to changes in cell cycle distribution (% S/G2 60.1% vs 63.2% for EV and E7, respectively). Conclusion: HPV16 E7 expression results in a defect in cNHEJ as well as upregulation of HR and altEJ. Human data from the TCGA is consistent with the observed increase in altEJ in HPV+ tumors. The increased utilization of altEJ in HPV+ tumors may contribute to the observed radiosensitivity by increasing error-prone lethal repair. Potential therapeutic targets and biomarkers for radiosensitization in HPV-positive cancers are suggested by this work. Author Disclosure: J.E. Leeman: None. R. Damerla: None. N. Riaz: None. N. Lee: Advisory Board; Merck, Pfizer, Vertex. S.N. Powell: None. D.S. Higginson: None.
Abstract 62; Table 1
Skewness (mean) Kurtosis (mean) Tumor Vol (mean)
0 weeks
6 weeks
12 weeks
0.87 1.65 290cc
-0.21 1.63 100cc
-0.23 1.28 80.6cc
Skewness of the distribution of voxels in the TVs between 0 and 6 weeks and 0 and 12 weeks were significantly different (P < 0.01) and less positive indicating a shift in distribution to a greater proportion of higher ADC values. This shift towards less restricted water movement may be consistent with development of tumor necrosis and less tumor cellularity. No other changes were significant. bGTVs generated at week 12 were distributed evenly between a more peripheral pattern of low ADC values (n Z 5) and more nodular mass-like pattern of low ADC values (n Z 5). Conclusion: ADC values exhibited an increasing trend during the initial 6-12 weeks of chemotherapy in children with newly diagnosed RMS consistent with necrosis in response to therapy. Areas of restricted diffusion, representing residual tumor cellularity, evolved to either a peripheral or nodular pattern and would be potential bGTVs. This trial will continue to collect DWI to better define these initial trends. Author Disclosure: M. Krasin: None. C. Hillenbrand: None. M. McCarville: None. D.J. Indelicato: Expert Witness; Mateer Harbert PA. Honoraria; Mayo Clinic, University of Cincinnati. Consultant; LEK Consulting, Maximus Federal Services. Travel Expenses; IBA; Pediatric Proton Foundation. J. Uh: None. A. Pappo: None. J. Lucas: None. C. Hua: None.
63 Upregulation of Alternative End Joining in HPV+ Head and Neck Cancer J.E. Leeman, R. Damerla, N. Riaz, N. Lee, S.N. Powell, and D.S. Higginson; Memorial Sloan Kettering Cancer Center, New York, NY Purpose/Objective(s): Head and neck squamous cell carcinomas (HNSCC) associated with the human papillomavirus (HPV) demonstrate remarkable radiosensitivity for reasons that are not entirely clear. HPV driven alterations in the DNA damage response may underlie the enhanced radiation response, though the specific repair pathways that are disrupted have not been elucidated. Alternative end joining (altEJ) is an error-prone double strand break (DSB) repair pathway that has been linked to radiosensitivity, but is less studied than other major DSB repair pathways (homologous recombination, HR, and non-homologous end joining, NHEJ), and has never been examined in the context of HPV-associated tumors. Materials/Methods: Genomic data from The Cancer Genome Atlas (TCGA) was used to assess proportions of deletions with surrounding microhomology (3-10 base pairs) as a measure of altEJ as well as expression of polymerase theta (POLQ), an essential component of the altEJ machinery. We utilized green fluorescent protein (GFP) based reporters of DSB repair pathways, including HR (DR-GFP), NHEJ (EJ5GFP) and altEJ (EJ2-GFP) to assess DSB repair pathway choice. Separate U2OS cell lines with integrated copies of each reporter system were transiently transfected with Isce1 endonuclease to generate DSBs with or without HPV16 E7 or the HPV16 E7 C24G mutant (defective in Rb
64 ATR Kinase Inhibition Leads to Durable Radiosensitization of the Murine KrasG12D/Twist1 Lung Adenocarcinoma Model via a CD8+ T CellDependent Mechanism P. Karukonda,1 F.P. Vendetti,1 P.T. Tran,2 D.A. Clump II3 R.L. Ferris,4 and C.J. Bakkenist5; 1University of Pittsburgh School of Medicine, Pittsburgh, PA, 2Johns Hopkins University School of Medicine, Department of Radiation Oncology and Molecular Radiation Sciences, Baltimore, MD, 3University of Pittsburgh Cancer Institute, Pittsburgh, PA, 4 University of Pittsburgh Medical Center, Pittsburgh, PA, 5University of Pittsburgh, Pittsburgh, PA Purpose/Objective(s): Ataxia Telangiectasia and Rad3-related (ATR) is a DNA-damage signaling kinase that is activated at damaged replication forks and resected double-strand breaks. ATR kinase inhibitors (ATRi’s) have been shown to increase the efficacy of cisplatin and ionizing radiation (IR) treatment of lung cancer by blocking DNA repair and cell cycle checkpoints. We hypothesize that ATRi’s radiosensitize lung cancer cells not only via cell cycle checkpoint blockade, but also via immune checkpoint inhibition in the form of PD-L1 downregulation. Materials/Methods: KrasG12D/Twist1 (CCSP-rtTA/tetO-KrasG12D/Twist1tetO7-luc) mice were administered doxycycline in their drinking water for 20-25 weeks to induce tumor development. The mice were randomized to study arms and treated with combinations of ATRi (75 mg/kg PO, q5d x 2), IR (hemi thoracic, 3 Gy x 5 x 2), and anti-CD8 antibody (250 ug IP, days 1, 4, 8, and 11). High resolution micro-CT scanning was done throughout the study to quantitate tumor burden. CT26 murine colon carcinoma cell line was used due to its Kras-mutant status and its documented immunogenicity. CRISPR-Cas9 mediated genome editing was used to create knockout (KO) cell lines. Anti PD-L1 antibody (clone 10F.9G2) and isotype were used at a concentration of 1:100 in flow cytometry experiments. Results: We show that ATRi in combination with hemi thoracic IR results in a sustained decrease in tumor volume over the course of 5 weeks, in comparison to control arms. Importantly, this effect is abrogated by CD8+ T cell depletion, highlighting the presence of an immunomodulatory drug
Volume 99 Number 2S Supplement 2017 effect. In vitro, using Kras-mutant CT26 cells, we discovered that ATRi (300 nM) attenuates an increase in cell surface PD-L1 expression seen 24 hours after IR treatment (2 Gy and 6 Gy), as well as interferon-gamma (IFN-y) treatment (50 U/mL). Using Annexin V/Propidium iodide assays, we show no difference between arms in the amount of cell death, suggesting that inhibition of PD-L1 expression by ATRi is not dependent on apoptotic signaling. Through the creation of p53 and IRF-1 KO cell lines, we additionally rule out the contributions of cell-intrinsic DNA damage signaling and type II interferon signaling. We then show that rapamycin, an mTOR inhibitor, attenuates PD-L1 upregulation after IR and IFN-y treatment. We will thus next focus our efforts on investigating the overlap between mTOR and ATR kinase signaling to determine the mechanism of this PD-L1 upregulation. Conclusion: With this work, we showcase our investigation into a novel potential mechanism for the efficacy of ATRi’s. ATRi’s are currently in Phase 1 clinical trials, which creates the necessity for an intricate understanding of their mechanisms as cancer therapeutics. Our current and future response and mechanistic data could be used to inform treatment regimens in trials created to maximize patient outcomes. Author Disclosure: P. Karukonda: None. F.P. Vendetti: None. P.T. Tran: Research Grant; NIH-NCI, ACS, Kimmel Foundation, Astellas-Medivation, American Lung Association, Movember-PCF, PCORI. Honoraria; Dendreon. Advisory Board; Astellas-Medivation, Dendreon. Travel Expenses; Dendreon. Patent/License Fees/Copyright; Compounds and Methods of Use in Ablative RT, Natsar Pharmaceuticals; RSNA R&E Foundation. D.A. Clump: None. R.L. Ferris: Employee; University of Pittsburgh Medical Center. Research Grant; BMS, VentiRX. Honoraria; BMS. Advisory Board; BMS. Travel Expenses; BMS; University of Pittsburgh School of Medicine. C.J. Bakkenist: None.
65 A KRAS-NRF2-53BP1 Nonhomologous End-Joining Repair Pathway Mediates Oncogenic KRAS Radioresistance T.M. Williams,1 L. Yang,2 A. Estrada-Bernal,3 M. Chatterjee,2 and R. Robb1; 1The Ohio State University Wexner Medical Center, Department of Radiation Oncology, Columbus, OH, 2The Ohio State University, Columbus, OH, 3The Ohio State University, Ohio, OH Purpose/Objective(s): KRAS activating mutations are driver mutations commonly found in tumors for which radiotherapy is routinely used, including lung, colorectal, and pancreatic cancer. Clinical and preclinical data supports that KRAS activating mutations foster radioresistance, but whether nuclear DNA repair mechanisms are altered in KRAS mutant cells is unclear. We hypothesize that KRAS mutations promote radioresistance through increased double-strand break repair mechanisms. Materials/Methods: We tested the effects of various KRAS mutations using homologous-recombined, isogenic cell lines (HCT116 or SW48) bearing wild-type or mutant KRAS (G12C, G12D, G12V, G13D). We evaluated the role of KRAS and downstream targets in radiation clonogenic, in vivo tumor growth, neutral comet, nuclear foci, cell cycle analysis, mitotic catastrophe, immunoblotting, chromatin immunoprecipitation (ChIP), RT-PCR, and functional DNA repair reporter assays. Results: We have previously shown that the presence of a single codon 12 or 13 KRAS point mutation induces radioresistance in vitro, and that a KRAS mutation induces marked radioresistance in vivo. KRAS mutant cells demonstrate enhanced ability to repair DNA damage, as measured by heightened comet tail resolution, accelerated kinetics of gamma H2A.X nuclear foci resolution, reduced mitotic catastrophe, and earlier escape from radiation-induced G2/M arrest after radiation (RT). In functional reporter assays, the presence of a KRAS mutation suppresses homology directed repair (HDR) repair, but promotes non-homologous end-joining (NHEJ) repair. We have identified that 53BP1, a critical intermediate of NHEJ repair, is up-regulated in KRAS mutant tumor cells, and that 53BP1 nuclear foci are rapidly induced in KRAS mutant cells after RT, and resolved earlier than KRAS wild-type cells. Moreover, 53BP1 depletion
Oral Scientific Sessions
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preferentially radiosensitizes KRAS mutant cells in vitro, and stable depletion effectively radiosensitizes tumors in vivo. We demonstrate that KRAS mutant cells also up-regulate Nrf2 (an antioxidant response transcription factor) and Nrf2-related transcriptional targets, and that KRAS genetic or chemical silencing reduces both 53BP1 and Nrf2 expression in KRAS mutant cells. Using ChIP, we demonstrate that Nrf2 binds to 53BP1 promoter elements to a greater extent in KRAS mutant cells. Furthermore, genetic or chemical suppression of Nrf2 reduces 53BP1 RNA and protein expression, and effectively radiosensitizes KRAS mutant cells, but not KRAS wild-type tumor cells. Conclusion: We have identified a KRAS-Nrf2-53BP1 oncogenic transcriptional regulatory pathway that imparts accelerated DNA repair and survival to KRAS mutant tumor cells after RT. These findings indicate that activating KRAS mutations recruit NHEJ pathways of DNA repair to more quickly repair double-strand breaks induced by RT. Targeting Nrf2 and 53BP1 may represent novel strategies to selectively abrogate KRAS mutant mediated radioresistance. Author Disclosure: T.M. Williams: Research Grant; Varian Medical Systems, Inc. Consultant; Covidien. Advisory Board; Covidien. I am the Gastrointestinal Cancers liaison; NRG Oncology.; National Cancer Institute. L. Yang: None. A. Estrada-Bernal: None. M. Chatterjee: None. R. Robb: None.
66 A CRISPR Screen to Systematically Identify DNA Repair Gene Alterations that Modulate Sensitivity to DNA Damaging Agents G.P. Gupta,1 A. Guo,2 W. Feng,2 and D.A. Simpson2; 1University of North Carolina, Chapel Hill, NC, 2University of North Carolina at Chapel Hill, Chapel Hill, NC Purpose/Objective(s): To implement a CRISPR loss of function screen to systematically identify DNA repair genes that are required for viability after exposure to different types of DNA damaging agents (DDAs). Materials/Methods: A lentiviral pooled DDR-CRISPR plasmid library was generated that targets 310 murine genes that are implicated in the DNA damage response, comprised of ten small guide RNAs (sgRNAs) designed to each gene and 900 additional non-targeting sgRNA controls. A SV40-transformed murine embryonic fibroblast (MEF) cell line was transduced with the DDR-CRISPR library at multiplicity of infection <1, antibiotic selected, and subsequently exposed to vehicle control, 1 Gy daily ionizing radiation, or IC50 doses of paclitaxel, camptothecin, cisplatin, mechlorethamine, or 5-fluorouracil. After six population doublings, genomic DNA was harvested, sgRNA sequences amplified, multiplexed, and analyzed by high throughput sequencing using an Ion Proton instrument. A bioinformatics pipeline was implemented to quantify relative abundance of each sgRNA sequence. Gene targeting sgRNAs that were significantly depleted or enriched in the presence of DDAs relative to control cells were identified using a P<0.05 threshold for an empirical null distribution defined by the control sgRNA sequences included in the DDRCRISPR screen. Validation experiments were performed using independent gene-targeting sgRNAs and colony forming assays after exposure to the DDA(s) of interest. Results: We identified 124 DNA damage response genes that, when disrupted by CRISPR targeting, significantly altered sensitivity to at least one of the six different types of DDAs evaluated in this study. Notably, only one gene (DKC1) significantly affected sensitivity to all six DDAs. In contrast, there were 72 genes that impacted sensitivity to only one out of six DDAs, indicating remarkable selectivity in the genes required for repair of distinct types of exogenous DNA damage. The validation rate of genetic associations identified in the DDR-CRISPR screen was >90% using independent sgRNAs and colony forming assays after DDA exposure. Analysis of TCGA datasets indicate that approximately 40% of all cancers harbor homozygous deletion or truncating mutations in at least one of the 124 genes identified in this study thatdwhen disrupteddmodulate sensitivity to DDAs.
International Journal of Radiation Oncology Biology Physics
thresholding the week 12 TV for ADC values below the week 0 mean ADC (theoretically representing the active cellular component of tumor at diagnosis). Results: The median ADC (average for TV) at 0, 6 and 12 weeks was 1.48x10-3, 1.54x10-3 and 1.65x10-3 mm2/s with a generally increasing trend for ADC. ADC values between weeks 0 and 6 were significantly different (P<0.01), while values between weeks 0 and 12 (P Z 0.06) and weeks 6 and 12 (P Z 0.13) were not significant. ADC distributions and TVs are shown below.
inhibition). For the EJ5-GFP NHEJ reporter, Isce1-Trex2 (exonuclease) was used to generate blunt-ended DSBs. Percentages of GFP+ cells were measured by flow cytometry. Propidium iodide staining was used to measure cell cycle distribution. Results: Compared to HPV- HNSCC, HPV+ cases demonstrated a 33% increase in the proportion of deletions with flanking microhomology (n Z 949 deletions, 28% HPV+ vs 21% HPV-, P Z 0.005). TCGA RNAseq data revealed mRNA expression of POLQ to be 2.2 fold higher in HPV+ vs. HPV- cases (95% CI 2.1-2.4, P<0.001). Among 33 histologies of TCGA solid tumors, HPV+ HNSCC demonstrated the highest expression of POLQ (2.20.18 fold increase from HPV- HNSCC, P<0.001); cervical cancers were second highest. Using reporter assays, compared to empty vector (EV), expression of HPV16 E7 resulted in a decrease in NHEJ (-34%8%, P Z 0.003), and increases in HR (+8933%, P Z 0.002) and altEJ (+49%20%, P<0.001). Expression of the HPV16 E7 C24G mutant demonstrated no change in NHEJ (+0.3%9%, P Z 0.98), HR (+1845%, P Z 0.39) or altEJ (+1526%, P Z 0.24). Differences in DSB pathway utilization were not related to changes in cell cycle distribution (% S/G2 60.1% vs 63.2% for EV and E7, respectively). Conclusion: HPV16 E7 expression results in a defect in cNHEJ as well as upregulation of HR and altEJ. Human data from the TCGA is consistent with the observed increase in altEJ in HPV+ tumors. The increased utilization of altEJ in HPV+ tumors may contribute to the observed radiosensitivity by increasing error-prone lethal repair. Potential therapeutic targets and biomarkers for radiosensitization in HPV-positive cancers are suggested by this work. Author Disclosure: J.E. Leeman: None. R. Damerla: None. N. Riaz: None. N. Lee: Advisory Board; Merck, Pfizer, Vertex. S.N. Powell: None. D.S. Higginson: None.
Abstract 62; Table 1
Skewness (mean) Kurtosis (mean) Tumor Vol (mean)
0 weeks
6 weeks
12 weeks
0.87 1.65 290cc
-0.21 1.63 100cc
-0.23 1.28 80.6cc
Skewness of the distribution of voxels in the TVs between 0 and 6 weeks and 0 and 12 weeks were significantly different (P < 0.01) and less positive indicating a shift in distribution to a greater proportion of higher ADC values. This shift towards less restricted water movement may be consistent with development of tumor necrosis and less tumor cellularity. No other changes were significant. bGTVs generated at week 12 were distributed evenly between a more peripheral pattern of low ADC values (n Z 5) and more nodular mass-like pattern of low ADC values (n Z 5). Conclusion: ADC values exhibited an increasing trend during the initial 6-12 weeks of chemotherapy in children with newly diagnosed RMS consistent with necrosis in response to therapy. Areas of restricted diffusion, representing residual tumor cellularity, evolved to either a peripheral or nodular pattern and would be potential bGTVs. This trial will continue to collect DWI to better define these initial trends. Author Disclosure: M. Krasin: None. C. Hillenbrand: None. M. McCarville: None. D.J. Indelicato: Expert Witness; Mateer Harbert PA. Honoraria; Mayo Clinic, University of Cincinnati. Consultant; LEK Consulting, Maximus Federal Services. Travel Expenses; IBA; Pediatric Proton Foundation. J. Uh: None. A. Pappo: None. J. Lucas: None. C. Hua: None.
63 Upregulation of Alternative End Joining in HPV+ Head and Neck Cancer J.E. Leeman, R. Damerla, N. Riaz, N. Lee, S.N. Powell, and D.S. Higginson; Memorial Sloan Kettering Cancer Center, New York, NY Purpose/Objective(s): Head and neck squamous cell carcinomas (HNSCC) associated with the human papillomavirus (HPV) demonstrate remarkable radiosensitivity for reasons that are not entirely clear. HPV driven alterations in the DNA damage response may underlie the enhanced radiation response, though the specific repair pathways that are disrupted have not been elucidated. Alternative end joining (altEJ) is an error-prone double strand break (DSB) repair pathway that has been linked to radiosensitivity, but is less studied than other major DSB repair pathways (homologous recombination, HR, and non-homologous end joining, NHEJ), and has never been examined in the context of HPV-associated tumors. Materials/Methods: Genomic data from The Cancer Genome Atlas (TCGA) was used to assess proportions of deletions with surrounding microhomology (3-10 base pairs) as a measure of altEJ as well as expression of polymerase theta (POLQ), an essential component of the altEJ machinery. We utilized green fluorescent protein (GFP) based reporters of DSB repair pathways, including HR (DR-GFP), NHEJ (EJ5GFP) and altEJ (EJ2-GFP) to assess DSB repair pathway choice. Separate U2OS cell lines with integrated copies of each reporter system were transiently transfected with Isce1 endonuclease to generate DSBs with or without HPV16 E7 or the HPV16 E7 C24G mutant (defective in Rb
64 ATR Kinase Inhibition Leads to Durable Radiosensitization of the Murine KrasG12D/Twist1 Lung Adenocarcinoma Model via a CD8+ T CellDependent Mechanism P. Karukonda,1 F.P. Vendetti,1 P.T. Tran,2 D.A. Clump II3 R.L. Ferris,4 and C.J. Bakkenist5; 1University of Pittsburgh School of Medicine, Pittsburgh, PA, 2Johns Hopkins University School of Medicine, Department of Radiation Oncology and Molecular Radiation Sciences, Baltimore, MD, 3University of Pittsburgh Cancer Institute, Pittsburgh, PA, 4 University of Pittsburgh Medical Center, Pittsburgh, PA, 5University of Pittsburgh, Pittsburgh, PA Purpose/Objective(s): Ataxia Telangiectasia and Rad3-related (ATR) is a DNA-damage signaling kinase that is activated at damaged replication forks and resected double-strand breaks. ATR kinase inhibitors (ATRi’s) have been shown to increase the efficacy of cisplatin and ionizing radiation (IR) treatment of lung cancer by blocking DNA repair and cell cycle checkpoints. We hypothesize that ATRi’s radiosensitize lung cancer cells not only via cell cycle checkpoint blockade, but also via immune checkpoint inhibition in the form of PD-L1 downregulation. Materials/Methods: KrasG12D/Twist1 (CCSP-rtTA/tetO-KrasG12D/Twist1tetO7-luc) mice were administered doxycycline in their drinking water for 20-25 weeks to induce tumor development. The mice were randomized to study arms and treated with combinations of ATRi (75 mg/kg PO, q5d x 2), IR (hemi thoracic, 3 Gy x 5 x 2), and anti-CD8 antibody (250 ug IP, days 1, 4, 8, and 11). High resolution micro-CT scanning was done throughout the study to quantitate tumor burden. CT26 murine colon carcinoma cell line was used due to its Kras-mutant status and its documented immunogenicity. CRISPR-Cas9 mediated genome editing was used to create knockout (KO) cell lines. Anti PD-L1 antibody (clone 10F.9G2) and isotype were used at a concentration of 1:100 in flow cytometry experiments. Results: We show that ATRi in combination with hemi thoracic IR results in a sustained decrease in tumor volume over the course of 5 weeks, in comparison to control arms. Importantly, this effect is abrogated by CD8+ T cell depletion, highlighting the presence of an immunomodulatory drug
Volume 99 Number 2S Supplement 2017 effect. In vitro, using Kras-mutant CT26 cells, we discovered that ATRi (300 nM) attenuates an increase in cell surface PD-L1 expression seen 24 hours after IR treatment (2 Gy and 6 Gy), as well as interferon-gamma (IFN-y) treatment (50 U/mL). Using Annexin V/Propidium iodide assays, we show no difference between arms in the amount of cell death, suggesting that inhibition of PD-L1 expression by ATRi is not dependent on apoptotic signaling. Through the creation of p53 and IRF-1 KO cell lines, we additionally rule out the contributions of cell-intrinsic DNA damage signaling and type II interferon signaling. We then show that rapamycin, an mTOR inhibitor, attenuates PD-L1 upregulation after IR and IFN-y treatment. We will thus next focus our efforts on investigating the overlap between mTOR and ATR kinase signaling to determine the mechanism of this PD-L1 upregulation. Conclusion: With this work, we showcase our investigation into a novel potential mechanism for the efficacy of ATRi’s. ATRi’s are currently in Phase 1 clinical trials, which creates the necessity for an intricate understanding of their mechanisms as cancer therapeutics. Our current and future response and mechanistic data could be used to inform treatment regimens in trials created to maximize patient outcomes. Author Disclosure: P. Karukonda: None. F.P. Vendetti: None. P.T. Tran: Research Grant; NIH-NCI, ACS, Kimmel Foundation, Astellas-Medivation, American Lung Association, Movember-PCF, PCORI. Honoraria; Dendreon. Advisory Board; Astellas-Medivation, Dendreon. Travel Expenses; Dendreon. Patent/License Fees/Copyright; Compounds and Methods of Use in Ablative RT, Natsar Pharmaceuticals; RSNA R&E Foundation. D.A. Clump: None. R.L. Ferris: Employee; University of Pittsburgh Medical Center. Research Grant; BMS, VentiRX. Honoraria; BMS. Advisory Board; BMS. Travel Expenses; BMS; University of Pittsburgh School of Medicine. C.J. Bakkenist: None.
65 A KRAS-NRF2-53BP1 Nonhomologous End-Joining Repair Pathway Mediates Oncogenic KRAS Radioresistance T.M. Williams,1 L. Yang,2 A. Estrada-Bernal,3 M. Chatterjee,2 and R. Robb1; 1The Ohio State University Wexner Medical Center, Department of Radiation Oncology, Columbus, OH, 2The Ohio State University, Columbus, OH, 3The Ohio State University, Ohio, OH Purpose/Objective(s): KRAS activating mutations are driver mutations commonly found in tumors for which radiotherapy is routinely used, including lung, colorectal, and pancreatic cancer. Clinical and preclinical data supports that KRAS activating mutations foster radioresistance, but whether nuclear DNA repair mechanisms are altered in KRAS mutant cells is unclear. We hypothesize that KRAS mutations promote radioresistance through increased double-strand break repair mechanisms. Materials/Methods: We tested the effects of various KRAS mutations using homologous-recombined, isogenic cell lines (HCT116 or SW48) bearing wild-type or mutant KRAS (G12C, G12D, G12V, G13D). We evaluated the role of KRAS and downstream targets in radiation clonogenic, in vivo tumor growth, neutral comet, nuclear foci, cell cycle analysis, mitotic catastrophe, immunoblotting, chromatin immunoprecipitation (ChIP), RT-PCR, and functional DNA repair reporter assays. Results: We have previously shown that the presence of a single codon 12 or 13 KRAS point mutation induces radioresistance in vitro, and that a KRAS mutation induces marked radioresistance in vivo. KRAS mutant cells demonstrate enhanced ability to repair DNA damage, as measured by heightened comet tail resolution, accelerated kinetics of gamma H2A.X nuclear foci resolution, reduced mitotic catastrophe, and earlier escape from radiation-induced G2/M arrest after radiation (RT). In functional reporter assays, the presence of a KRAS mutation suppresses homology directed repair (HDR) repair, but promotes non-homologous end-joining (NHEJ) repair. We have identified that 53BP1, a critical intermediate of NHEJ repair, is up-regulated in KRAS mutant tumor cells, and that 53BP1 nuclear foci are rapidly induced in KRAS mutant cells after RT, and resolved earlier than KRAS wild-type cells. Moreover, 53BP1 depletion
Oral Scientific Sessions
S31
preferentially radiosensitizes KRAS mutant cells in vitro, and stable depletion effectively radiosensitizes tumors in vivo. We demonstrate that KRAS mutant cells also up-regulate Nrf2 (an antioxidant response transcription factor) and Nrf2-related transcriptional targets, and that KRAS genetic or chemical silencing reduces both 53BP1 and Nrf2 expression in KRAS mutant cells. Using ChIP, we demonstrate that Nrf2 binds to 53BP1 promoter elements to a greater extent in KRAS mutant cells. Furthermore, genetic or chemical suppression of Nrf2 reduces 53BP1 RNA and protein expression, and effectively radiosensitizes KRAS mutant cells, but not KRAS wild-type tumor cells. Conclusion: We have identified a KRAS-Nrf2-53BP1 oncogenic transcriptional regulatory pathway that imparts accelerated DNA repair and survival to KRAS mutant tumor cells after RT. These findings indicate that activating KRAS mutations recruit NHEJ pathways of DNA repair to more quickly repair double-strand breaks induced by RT. Targeting Nrf2 and 53BP1 may represent novel strategies to selectively abrogate KRAS mutant mediated radioresistance. Author Disclosure: T.M. Williams: Research Grant; Varian Medical Systems, Inc. Consultant; Covidien. Advisory Board; Covidien. I am the Gastrointestinal Cancers liaison; NRG Oncology.; National Cancer Institute. L. Yang: None. A. Estrada-Bernal: None. M. Chatterjee: None. R. Robb: None.
66 A CRISPR Screen to Systematically Identify DNA Repair Gene Alterations that Modulate Sensitivity to DNA Damaging Agents G.P. Gupta,1 A. Guo,2 W. Feng,2 and D.A. Simpson2; 1University of North Carolina, Chapel Hill, NC, 2University of North Carolina at Chapel Hill, Chapel Hill, NC Purpose/Objective(s): To implement a CRISPR loss of function screen to systematically identify DNA repair genes that are required for viability after exposure to different types of DNA damaging agents (DDAs). Materials/Methods: A lentiviral pooled DDR-CRISPR plasmid library was generated that targets 310 murine genes that are implicated in the DNA damage response, comprised of ten small guide RNAs (sgRNAs) designed to each gene and 900 additional non-targeting sgRNA controls. A SV40-transformed murine embryonic fibroblast (MEF) cell line was transduced with the DDR-CRISPR library at multiplicity of infection <1, antibiotic selected, and subsequently exposed to vehicle control, 1 Gy daily ionizing radiation, or IC50 doses of paclitaxel, camptothecin, cisplatin, mechlorethamine, or 5-fluorouracil. After six population doublings, genomic DNA was harvested, sgRNA sequences amplified, multiplexed, and analyzed by high throughput sequencing using an Ion Proton instrument. A bioinformatics pipeline was implemented to quantify relative abundance of each sgRNA sequence. Gene targeting sgRNAs that were significantly depleted or enriched in the presence of DDAs relative to control cells were identified using a P<0.05 threshold for an empirical null distribution defined by the control sgRNA sequences included in the DDRCRISPR screen. Validation experiments were performed using independent gene-targeting sgRNAs and colony forming assays after exposure to the DDA(s) of interest. Results: We identified 124 DNA damage response genes that, when disrupted by CRISPR targeting, significantly altered sensitivity to at least one of the six different types of DDAs evaluated in this study. Notably, only one gene (DKC1) significantly affected sensitivity to all six DDAs. In contrast, there were 72 genes that impacted sensitivity to only one out of six DDAs, indicating remarkable selectivity in the genes required for repair of distinct types of exogenous DNA damage. The validation rate of genetic associations identified in the DDR-CRISPR screen was >90% using independent sgRNAs and colony forming assays after DDA exposure. Analysis of TCGA datasets indicate that approximately 40% of all cancers harbor homozygous deletion or truncating mutations in at least one of the 124 genes identified in this study thatdwhen disrupteddmodulate sensitivity to DDAs.