Radiosensitization of Glioblastoma Stem Cells through Targeting Metabolic Reprogramming

Volume 96  Number 2S  Supplement 2016 higher expression levels of the ALDH1A1, EpCAM, and CXCR4. Tumor sphere formation reduced dramatically in CR cells following genetic knockdown of Stat3 (11.5  1.5 vs 4.8  1.1, P < 0.001) or exposure to S3I-201. Furthermore, when viable tumor spheres were dissociated and replated in the absence of the drug, a significant impact on secondary tumor sphere formation was also observed. CSC like cells contributes to radioresistance through preferential activation of the DNA damage checkpoint response. We found activating phosphorylation of checkpoint proteins ATM, CHK1 and CHK2 were significantly higher in CR cells than in NA cells indicating that CR cells show greater checkpoint activation in response to DNA damage. Consistent with CSC phenotype, H3122 CR showed more resistance to irradiation compare to the NA cells by clonogenic assay. Of note, the resistance can be abrogated by genetic silencing of Stat3. Because stat3 mediates transcription of INF-g and PD-L1, we found PD-L1 expression H3122 cell lines were modulated by Stat3 pathway. Ablation of Stat3 expression by Stat3 siRNA significantly (P < 0.001) impaired the higher PD-L1 expression in H3122 CR cells. Conclusion: Stat3 activation plays crucial role in modulating CSC-like properties, DNA repair, and immunosuppression in crizotinib resistance of ELM4-ALK-rearranged lung cancers. It provides a potential therapeutic target for this group of patients. Author Disclosure: W. Guo: None. S. Du: None. L. Yang: None. A.P. Dicker: None. B. Lu: None.

173 MIR384, Inhibited By NF-Kb, Enhances Radiosensitivity in Human Non-Small Cell Lung Cancer Via Modulating DNA Damage Response and Repair Signaling M. Qiu,1 Z. Wu,1 Z. Yuan,2 and M. Meng1; 1Department of Radiotherapy, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China, 2Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China Purpose/Objective(s): Radiotherapy achieved remarkable effects in the treatment of non-small cell lung cancer (NSCLC). However, radioresistance is still the major obstacles that decrease the outcomes of NSCLC patients. DNA damage response and repair signaling have been demonstrated greatly involving in modulating radiosensitivity and prognosis of patients with NSCLC. This study aims at identifying novel DDR signaling regulators and potential target for enhancing radiosensitivity of NSCLC. Materials/Methods: Lentivirus-based infection and CRISPR/Cas9 editing technology were applied for overexpression and depletion of microRNA384 (miR-384), respectively. Clonogenic assay and g-H2AX immunofluorescence staining were performed to analyze the effects of miR-384 on radiosensitivity of NSCLC cells. FACS was applied to detect cell death. Luciferase reporter assay, western blotting, and qRT-PCR were applied to prove ATM, Ku70, and Ku80 to be direct targets of miR-384. Luciferase reporter assay, Ch-IP, and qRT-PCR were applied to demonstrate that NF-kB inhibits miR-384 directly. Results: In the current study, we found that the expression of miR-384 positively correlated with radiosensitivity of NSCLC cells. Ectopic overexpression of miR-384 radiosensitized NSCLC cells by inhibiting DNA damage repair and increasing apoptosis, while knockout miR-384 led to radiation resistance with enhanced DNA damage repair and decreased apoptosis. Further investigation revealed that ATM, Ku70, and Ku80 were direct targets of miR-384. Moreover, we identified miR-384 to be a direct downstream gene of NF-kB, and NF-kB inhibits the expression of miR-384. Conclusion: In the current study, miR-384 was proved to enhance radiosensitivity via NF-kB/MIR384/ATM feedback loop and inhibiting Ku70 and Ku80 to impair DDR signaling in NSCLC. Here, we specifically highlighted a novel mechanism that microRNA involved in modulating DDR signaling and radiosensitivity of NSCLC. This study evidenced that miR-384 could be a novel therapeutic target for NSCLC. Author Disclosure: M. Qiu: None. Z. Wu: None. Z. Yuan: None. M. Meng: None.

Oral Scientific Sessions

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174 A Novel Breast Cancer Xenograft Model Identifies Genetic Variants in the Tumor Microenvironment that Enhance Radiation Responses M.W. Straza, A. Rymaszewski, A. Frei, A. Lemke, S.W. Tsaih, M. Flister, and C. Bergom; Medical College of Wisconsin, Milwaukee, WI Purpose/Objective(s): Communication between malignant tumor cells and the tumor microenvironment (TME) underlies many aspects of tumor biology, including radiation resistance. We have developed a Consomic Xenograft Model (CXM), which maps germline variants that impact only the TME, as well as a species-specific RNA-seq (SSRS) protocol which allows detection of expression changes in the malignant and nonmalignant cellular compartments of tumor xenografts, in parallel and without cellsorting. Here we utilize these novel techniques to identify genetic variants in the TME that affect radiation sensitivity Materials/Methods: Human triple negative breast cancer MDA-MD-231 cells were implanted into immunodeficient (IL2Rg-/-) consomic rat strains that are genetically identical except for chromosome 3 (SS and SS.BN3 strains). On day 10, tumors were either mock treated or treated locally with 3 daily ionizing radiation (IR) treatments of 4 Gy. Tumor growth was monitored and time to tumor recurrence assessed using the Kaplan Meier method. Separately, tumors were treated with mock or IR and harvested 24 hours after the last IR dose. RNA was purified, individual libraries prepped and indexed for multiplexing, and RNA-Seq was performed. A custom SSRS protocol was used to align both rat and human transcripts. This yielded transcript and gene level estimated fold-change and adjusted P values for human- and rat-derived transcripts separately. Chi-square, Fisher’s exact, and Kolmogorov-Smirnov tests and empirical cumulative distribution plots for differential expression significance values were performed. Results: Using CXM, we discovered that BN strain-derived genetic variant(s) on rat chromosome 3 are important for tumor IR sensitivity, as human breast cancer xenografts in the consomic strain (SS.BN3) are significantly more IR sensitive than tumors in the parental SS rat strain. A supra-additive difference was seen in the time to 5-fold increase in mean tumor size, with 44 vs. >130 days for radiated SS versus SS.BN3 rats. There was a recurrence-free survival of 30% vs. 67% at 130 days, with a median time to recurrence of 57 days vs. time not reached (>130 days) in the SS versus SS.BN3 rats (P Z 0.02). We also demonstrated that the transcriptional response to IR has surprisingly large differences between the tumor and stromal cells in xenografts, both in the number of genes differentially expressed and in the functional pathways altered. Conclusion: These results demonstrate that genetic determinants in the TME affect IR sensitivity of genetically identical tumor cells. Using SSRS, we identified candidate genes on rat chromosome 3 that may potentially influence IR sensitivity. Future studies will determine the pathways responsible for the changes in IR sensitivity. Determining TME factors that affect the IR sensitivity of tumors may allow for more tailored and effective treatment of breast cancer. These results demonstrate that CXM and SSRS are powerful tools to assess the TME impact on IR responses. Author Disclosure: M.W. Straza: None. A. Rymaszewski: student; Medical College of Wisconsin. A. Frei: None. A. Lemke: None. S. Tsaih: None. M. Flister: None. C. Bergom: None.

175 Radiosensitization of Glioblastoma Stem Cells through Targeting Metabolic Reprogramming K. Yang,1,2 X. Wang,2 Q. Xie,2 L.J.Y. Kim,2 W.A. Flavahan,2 S.T. Chao,3 and J.N. Rich2; 1Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, 2Department of Stem Cell Biology and Regenerative Medicine, Cleveland Clinic, Cleveland, OH, 3 Department of Radiation Oncology, Cleveland Clinic, Cleveland, OH Purpose/Objective(s): Glioblastoma (GBM) is a deadly form of brain tumor for which conventional treatments including radiation therapy are not curative. Metabolic reprogramming, namely the Warburg effect, is recognized as a key hallmark of cancer. Metabolic reprograming in cancer

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International Journal of Radiation Oncology  Biology  Physics

is not simply a passenger in tumorigenesis, but may be an initiating event as recurrent somatic mutations of metabolic enzymes have been reported. We showed previously that glioblastoma stem cells (GSCs), a subset of tumor cells that exhibit radiation resistance, hijack the process of highaffinity glucose uptake normally active in brain to maintain energy demands in dynamic tumor microenvironments. Here, we aim to understand the molecular mechanism of such aberrant metabolic pattern in GSCs and develop targeted approach to achieve radiation sensitization. Materials/Methods: GSCs and matched differentiated glioblastoma cells (DGCs) were derived from patient specimens and functionally validated. Matched pairs of GSCs and DGCs were treated with U-13C-glucose, followed by non-targeted small molecule mass spectrometry to identify metabolites with higher relative 13C incorporation in GSCs, which were then confirmed with targeted HPLC/mass spectrometry. Genetic validation of identified metabolic pathways was performed using TCGA GBM dataset. Relative expression between GSCs and DGCs were assessed using qRTPCR and Western blotting. Functional validation of target gene was performed in vitro for GSC viability (ATP-based assay) and self-renewal (neurosphere formation), and in vivo for tumorigenicity (orthotopic xenograft model). Radiation sensitization was performed using Cs-137 irradiator. Results: We showed that GSCs upregulate GLUT3, a high-affinity glucose transporter, to compete for glucose in response to nutrient supply fluctuations. GLUT3 mediated glucose influx regulates GSC maintenance and tumorigenicity. Inhibiting GLUT3 with RNA interference decreased GSC survival and sensitized to radiation. Using unbiased metabolomics analysis, we traced carbon flow following glucose influx into GSCs, and discovered downstream glucose metabolism pathways including de novo serine synthesis were functionally upregulated, mediating glucose-sustained anabolic metabolism. Inhibiting downstream carbon processing through RNA interference and pharmacologic inhibitors attenuated GSC viability after radiation, supporting metabolic reprogramming as potential therapeutic point of fragility. Elevated expression of downstream carbon processing enzymes predicts poor prognosis in GBM patients. Upstream transcriptional activation of metabolic reprogramming is mediated by MYC, a key transcription factor in GSC biology and cellular metabolism. Conclusion: Our results suggest that a stem-like state in GBM is associated with metabolic reprogramming to fuel the tumor hierarchy, revealing potential GSC cancer dependencies amenable to targeted therapy for radiation sensitization. Author Disclosure: K. Yang: None. X. Wang: None. Q. Xie: None. L.J. Kim: None. W.A. Flavahan: None. S.T. Chao: None. J.N. Rich: Head of department; Cleveland Clinic.

assays were used to quantify radiation sensitivity. The gH2AX and 53BP1 foci, as well as neutral comet assays were used to estimate DNA DSB repair. For 4 consecutive days mice bearing HT29 xenografts received i.p. injections of either solvent control or VJ115. Thirty mins later tumors were administered 0 or 2.2 Gy. On day 5, tumors were either injected with pimonidazole and processed for IHC or administered a top-up dose of either 20 or 30 Gy. Survival was quantified for 90 days. Animals were euthanized if tumor volume exceeded IACUC guidelines or if the mouse exhibited signs of distress. Results: Pharmacological or shRNA-mediated suppression of ENOX1 radiosensitized HUVECs. HT29 cell survival was independent of the VJ115 inhibitor, consistent with the fact that these cells do not express ENOX1. Quantification of neutral comet tail moment indicated that targeting ENOX1 impaired repair of DNA DSBs in HUVECs. Repair inhibition could be attributed, in part, to suppression of PARP1 activity and potentially dysfunctional recruitment of 53BP1 to sites of DSBs. The life span of non-irradiated tumor-bearing mice did not exceed 35 days. Survival 90 days after treatment of mice administered DMSO, 2.2 Gy q.d. x 4, and top-up of doses of 20 or 30 Gy was 20% and 40%, respectively. In contrast, survival of mice administered VJ115, 2.2 Gy q.d. x 4, and top-up doses of 20 or 30 Gy was 40% and 80%, respectively. The 30 Gy top-up dose revealed that 4 daily VJ115 injections increased relative survival by 50% compared to solvent control treated mice (from 40% to 80%; P < 0.01). This increase in survival was not due to a decrease in tumor hypoxia, as there were no differences in tumor hypoxia between treatment groups (P > 0.05). Conclusion: These results support the hypothesis that ENOX1 is potentially a therapeutic target for radiosensitization of tumor vasculature. Author Disclosure: C.A. Smith: None. K. Sekhar: None. P. Crooks: None. G.L. Traver: None. M.L. Freeman: None.

176 Targeting ENOX1 Impairs Repair of DNA DSBs in Endothelial Cells and Increases Survival of Tumor-Bearing Mice C.A. Smith,1 K. Sekhar,1 P. Crooks,2 G.L. Traver,1 and M.L. Freeman1; 1 Department of Radiation Oncology, Vanderbilt University Medical Center, Nashville, TN, 2University of Arkansas for Medical Sciences, Little Rock, AR Purpose/Objective(s): ENOX1 is an NADH oxidase that also exhibits disulfide-thiol exchange activity. It is expressed in normal tissue but not in many tumor cells such as HT29 colorectal adenocarcinoma cells. Previously we have shown that ENOX1 supports both vasculogenesis and tumor neoangiogenesis. Targeting ENOX1 has been shown to radiosensitize endothelial cells and tumor vasculature, thereby increasing radiationinduced tumor growth delay. However, it is unclear whether tumor growth delay translates into increased survival. Herein, we tested the hypothesis that targeting ENOX1 specifically in tumor stroma would lower the dose of radiation required to increase survival. Materials/Methods: HUVECs and HT29 cells were obtained from Cambrex Bio Science and ATCC respectively. ENOX1 was targeted using shRNA expressed from retrovirus or inhibited by the small molecule VJ115. Irradiation was performed using either a Cesium 137 irradiator (2.1 Gy/min) or 300 kVp/10 mA x-rays at 1.55 Gy/min. Colony formation

177 Volumetric Assessment of Tumor Vessel Interaction Is a Predictor of Surgical Candidacy in Patients With Borderline Resectable and Locally Advanced Pancreatic Cancer Treated With Stereotactic Body Radiation Therapy Z. Cheng,1 L.M. Rosati,2 L. Chen,1 O.Y. Mian,3 A. Narang,1 Y. Cao,4 J. Moore,1 S.P. Robertson,3 A. Hacker-Prietz, PA-C,2 J.M. Herman,2 and T.R. McNutt1; 1Johns Hopkins University School of Medicine, Baltimore, MD, 2Johns Hopkins University School of Medicine, Department of Radiation Oncology & Molecular Radiation Sciences, Baltimore, MD, 3Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, 4Johns Hopkins University, Baltimore, MD Purpose/Objective(s): Surgical resection offers the only chance of cure for non-metastatic pancreatic cancer; however, numerous factors must be taken into account such as tumor size, location, and proximity of the tumor to critical vascular structures. Stereotactic body radiation therapy (SBRT) may improve the likelihood of an unresectable patient to undergo surgery. We developed a method to improve the characterization of tumor location relative to critical vascular structure(s) that affects surgical resectability. Materials/Methods: Oncospace is a local learning health system that systematically captures clinical outcomes and radiotherapy treatment parameters and can generate overlap volume histograms (OVH)ea measure of spatial relationships between two structures. Minimum distances between the target volume and organs at risk (OAR) based on OVH, GTV, PTV, anatomic location by ICD-9 code, and resected status were queried. Normalized distance from PTV to both kidneys was calculated to determine tumor location within the pancreas. The shape relationship of the GTV to superior mesenteric (SMA), celiac, and common hepatic (CHA) arteries was characterized by distance of involvement (cm), target volume overlap into critical arteries (cc), and radiographic degree of involvement (no involvement, abutment, encasement, and occlusion). Each tumor was further characterized as SMA-dominant or celiac-dominant. Spatial relationships were validated against borderline resectable (BR) and locally