Intrinsic Radiation Sensitivity Research Articles

Gene expression signature predicts radiation sensitivity in cell lines using the integral of dose-response curve.

Although substantial efforts have been made to build molecular biomarkers to predict radiation sensitivity, the ability to accurately stratify the patients is still limited. In this study, we aim to leverage large-scale radiogenomics datasets to build genomic predictors of radiation response using the integral of the radiation dose-response curve. Two radiogenomics datasets consisting of 511 and 60 cancer cell lines were utilized to develop genomic predictors of radiation sensitivity. The intrinsic radiation sensitivity, defined as the integral of the dose-response curve (AUC) was used as the radioresponse variable. The biological determinants driving AUC and SF2 were compared using pathway analysis. To build the predictive model, the largest and smallest datasets consisting of 511 and 60 cancer cell lines were used as the discovery and validation cohorts, respectively, with AUC as the response variable. Utilizing a compendium of three pathway databases, we illustrated that integral of the radiobiological model provides a more comprehensive characterization of molecular processes underpinning radioresponse compared to SF2. Furthermore, more pathways were found to be unique to AUC than SF2-30, 288 and 38 in KEGG, REACTOME and WIKIPATHWAYS, respectively. Also, the leading-edge genes driving the biological pathways using AUC were unique and different compared to SF2. With regards to radiation sensitivity gene signature, we obtained a concordance index of 0.65 and 0.61 on the discovery and validation cohorts, respectively. We developed an integrated framework that quantifies the impact of physical radiation dose and the biological effect of radiation therapy in interventional pre-clinical model systems. With the availability of more data in the future, the clinical potential of this signature can be assessed, which will eventually provide a framework to integrate genomics into biologically-driven precision radiation oncology.

Characterization of Intrinsic Radiation Sensitivity in a Diverse Panel of Normal, Cancerous and CRISPR-Modified Cell Lines.

Intrinsic radiosensitivity is a major determinant of radiation response. Despite the extensive amount of radiobiological data available, variability among different studies makes it very difficult to produce high-quality radiosensitivity biomarkers or predictive models. Here, we characterize a panel of 27 human cell lines, including those derived from lung cancer, prostate cancer, and normal tissues. In addition, we used CRISPR-Cas9 to generate a panel of lines with known DNA repair defects. These cells were characterised by measuring a range of biological features, including the induction and repair of DNA double-strand breaks (DSBs), cell cycle distribution, ploidy, and clonogenic survival following X-ray irradiation. These results offer a robust dataset without inter-experimental variabilities for model development. In addition, we used these results to explore correlations between potential determinants of radiosensitivity. There was a wide variation in the intrinsic radiosensitivity of cell lines, with cell line Mean Inactivation Doses (MID) ranging from 1.3 to 3.4 Gy for cell lines, and as low as 0.65 Gy in Lig4-/- cells. Similar substantial variability was seen in the other parameters, including baseline DNA damage, plating efficiency, and ploidy. In the CRISPR-modified cell lines, residual DSBs were good predictors of cell survival (R2 = 0.78, p = 0.009), as were induced levels of DSBs (R2 = 0.61, p = 0.01). However, amongst the normal and cancerous cells, none of the measured parameters correlated strongly with MID (R2 < 0.45), and the only metrics with statistically significant associations are plating efficiency (R2 = 0.31, p = 0.01) and percentage of cell in S phase (R2 = 0.37, p = 0.005). While these data provide a valuable dataset for the modelling of radiobiological responses, the differences in the predictive power of residual DSBs between CRISPR-modified and other subgroups suggest that genetic alterations in other pathways, such as proliferation and metabolism, may have a greater impact on cellular radiation response. These pathways are often neglected in response modelling and should be considered in the future.

Evaluation of Radiation Sensitivity Differences in Mouse Liver Tumor Organoids Using CRISPR/Cas9-Mediated Gene Mutation.

To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1. The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vectors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay. The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) staining, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations. This study developed a radiation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.

Recent Advances in Electron Microscopy of Carbohydrate Nanoparticles.

Carbohydrate nanoparticles, both naturally derived and synthetic ones, have attracted scientific and industrial attention as high-performance renewable building blocks of functional materials. Electron microscopy (EM) has played a central role in investigations of their morphology and molecular structure, although the intrinsic radiation sensitivity of carbohydrate crystals has often hindered the in-depth characterization with EM techniques. This contribution reviews the recent advances in the electron microscopy of the carbohydrate nanoparticles. In particular, we highlight the recent efforts made to understand the three-dimensional shape and structural heterogeneity of nanoparticles using low-dose electron tomography and electron diffraction techniques coupled with cryogenic transmission electron microscopy.

Development and validation of genomic predictors of radiation sensitivity using preclinical data

BackgroundRadiation therapy is among the most effective and commonly used therapeutic modalities of cancer treatments in current clinical practice. The fundamental paradigm that has guided radiotherapeutic regimens are ‘one-size-fits-all’, which are not in line with the dogma of precision medicine. While there were efforts to build radioresponse signatures using OMICS data, their ability to accurately predict in patients is still limited.MethodsWe proposed to integrate two large-scale radiogenomics datasets consisting of 511 with 23 tissues and 60 cancer cell lines with 9 tissues to build and validate radiation response biomarkers. We used intrinsic radiation sensitivity, i.e., surviving fraction of cells (SF2) as the radiation response indicator. Gene set enrichment analysis was used to examine the biological determinants driving SF2. Using SF2 as a continuous variable, we used five different approaches, univariate, rank gene ensemble, rank gene multivariate, mRMR and elasticNet to build genomic predictors of radiation response through a cross-validation framework.ResultsThrough the pathway analysis, we found 159 pathways to be statistically significant, out of which 54 and 105 were positively and negatively enriched with SF2. More importantly, we found cell cycle and repair pathways to be enriched with SF2, which are inline with the fundamental aspects of radiation biology. With regards to the radiation response gene signature, we found that all multivariate models outperformed the univariate model with a ranking based approach performing well compared to other models, indicating complex biological processes underpinning radiation response.ConclusionTo summarize, we found biological processes underpinning SF2 and systematically compared different machine learning approaches to develop and validate predictors of radiation response. With more patient data available in the future, the clinical value of these biomarkers can be assessed that would allow for personalization of radiotherapy.

The dopamine receptor antagonist trifluoperazine prevents phenotype conversion and improves survival in mouse models of glioblastoma

Glioblastoma (GBM) is the deadliest adult brain cancer, and all patients ultimately succumb to the disease. Radiation therapy (RT) provides survival benefit of 6 mo over surgery alone, but these results have not improved in decades. We report that radiation induces a glioma-initiating cell phenotype, and we have identified trifluoperazine (TFP) as a compound that interferes with this phenotype conversion. TFP causes loss of radiation-induced Nanog mRNA expression, and activation of GSK3 with consecutive posttranslational reduction in p-Akt, Sox2, and β-catenin protein levels. TFP did not alter the intrinsic radiation sensitivity of glioma-initiating cells (GICs). Continuous treatment with TFP and a single dose of radiation reduced the number of GICs in vivo and prolonged survival in syngeneic and patient-derived orthotopic xenograft (PDOX) mouse models of GBM. Our findings suggest that the combination of a dopamine receptor antagonist with radiation enhances the efficacy of RT in GBM by preventing radiation-induced phenotype conversion of radiosensitive non-GICs into treatment-resistant, induced GICs (iGICs).

Comparable radiation response of ex vivo and in vivo irradiated tumor samples determined by residual γH2AX

Purposea) To investigate if an ex vivo cultured and irradiated tumor biopsy reflects and predicts the radiation response of the corresponding in vivo irradiated tumor measured with the DNA double strand break marker γH2AX foci. Materials and methodsFive human head and neck squamous cell carcinoma (hHNSCC) xenograft models were used. Fine needle biopsies were taken from anesthetized tumor-bearing NMRI nude mice prior to in vivo single dose irradiation (0, 2, 4, or 8 Gy) under ambient blood flow. Biopsies were ex vivo reoxygenated and irradiated with equivalent doses. Tumors and biopsies were fixed 24 h post irradiation, and γH2AX foci were assessed in oxygenated tumor regions. ResultsLinear regression analysis showed comparable slopes of the residual γH2AX foci dose–response curves in four out of five hHNSCC models when in vivo and ex vivo cohorts were compared. The slopes from ex vivo biopsies and in vivo tumors could classify the respective tumor model as sensitive or resistant according to the intrinsic radiation sensitivity (TCD50). ConclusionThe ability of ex vivo irradiated tumor biopsies to reflect and predict the intrinsic radiation response of in vivo tumors increases the translational potential of the ex vivo γH2AX foci assay as a diagnostic tool for clinical practice.

Impact of Radiation on Tumor Derived Exosome Production, Proteomics and Biodistribution

Exosomes are cell derived nanoparticles implicated in malignant processes, including the determination of organ specific metastasis. This study aims to test the hypothesis that radiation interferes with the ability of tumor-derived exosomes to establish a pre-metastatic niche by affecting exosome production and proteomics. To understand the effect of radiation across tumors with different radiation sensitivities, breast cancer cell lines with known organotropism (MDA-MB-231 parental and the lung tropic subline, 4175) were compared to melanoma cancer cell line (B16F10) and exposed to 2 Gy vs. 10 Gy radiation. Exosomes were harvested from conditioned media using differential centrifugation and characterized using protein BCA, NanoSight, and electron microscopy. Exosome size was further characterized using a novel asymmetric flow fractionator. DNA packaging was quantified with Qubit ds DNA assay kit and proteomics analyzed through mass spectrometry. Biodistribution was assessed in vivo through retro-orbital sinus injection of fluorescently labeled exosomes and subsequent imaging of organ distribution. Radiation increased the quantity of exosomes produced in a dose dependent manner. Across all cell lines, 2 Gy vs. 10 Gy radiation increased exosome yield (ug/cell) by an average of 2.1 ± 0.4 and 7.8 ± 2.7-fold, respectively. In radioresistant melanoma cells, 2 Gy and 10 Gy radiation increased DNA content per exosome by 1.9 and 8.2-fold, respectively, whereas in more radiosensitive breast cancer cells, 2 Gy and 10 Gy only moderately increased DNA packaging by 1.1 and 1.4-fold. In general, radiation decreased exosome particle size. In breast cancer cells, mean particle size decreased from 135±6.9 nm to 125.7±1.8 and 124.3±3.9 nm for 2 Gy and 10 Gy radiation, respectively. Radiation also decreased the abundance of the smallest exosome subparticles, exomeres, which preferentially traffic to the liver to effect changes in metabolomics. For breast cancer cell lines, radiated exosomes retained the ability to traffic to the lung and liver 24 hours after injection. Pathway analysis of proteomic changes across radiation subgroups implicate significant changes in integrin signaling and remodeling of epithelial adherens junctions, including integrins and metalloproteases. Radiation significantly increased the quantity and DNA packaging of exosomes across all tumor cell lines. Although radiated exosomes trafficked to potential metastatic sites 24 hours after injection, their function in establishing a pre-metastatic niche remains to be explored. Changes in proteomics and exosome subpopulations suggest differences in migration and liver education. These changes trended with radiation dose and intrinsic radiation sensitivity, offering a promising platform for monitoring treatment response and underlying tumor biology in patients.

Abstract P1-09-05: Radiosensitization of androgen receptor (AR)-positive triple-negative breast cancer (TNBC) cells using seviteronel (INO-464), a selective CYP17 lyase and AR inhibitor

Abstract Background: Increased rates of locoregional recurrence (LR) have been observed in TNBC despite chemotherapy and radiation (RT). A novel radiosensitizer screen nominated the AR as a promising target for treatment of radioresistant breast cancer, including TNBC. We assessed the activity of seviteronel (Sevi), a selective CYP17 lyase and AR inhibitor in Phase 2 clinical development for advanced breast and prostate cancer, as a potential radiosensitizer in AR+ TNBC model. Methods: Clonogenic survival assays were used to determine the intrinsic RT sensitivity of 21 breast cancer cell (BCC) lines. IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Gene expression was measured using RNA Seq or qRT-PCR and protein expression was measured using RPPA arrays. AR function was assessed using functional inhibition with Sevi in MDA-MB-453, ACC-422, ACC-460, SUM-185 (all four AR+ TNBC), MDA-MB-231 (AR- TNBC), and T47D (AR- ER+) BCC lines. Double-stranded DNA (dsDNA) break repair was assessed with γH2AX foci counting. In vivo tumor growth was measured with varying control and treatment groups (16-20 tumors/group). Kaplan-Meier analysis was performed to estimate local control. A Cox proportional hazards model and multi-variate analysis (MVA) were used to determine variables associated with LRF survival. Results: Our novel radiosensitizer screen identified the activity of anti-androgen therapy as a potentially effective strategy for radiosensitization in RT-resistant BCC lines (R2 =0.46, p-value &amp;lt; 0.01) (Speers et al, J Clin Oncol 35, 2017 (suppl; abstr e12102). Heterogeneity in AR expression was identified in human BCC lines and TNBC samples from patients (N=2098). There was a strong correlation between AR RNA expression and protein expression across all BC intrinsic subtypes. AR inhibition using Sevi induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.24-1.69 in four different AR+ TNBC lines. No such radiosensitization was seen in AR(-) TNBC or ER+, AR(-) BCC lines. Radiosensitization was at least partially dependent on impaired dsDNA break repair with significant delays in dsDNA break repair at 16 and 24 hours in all AR+ TNBC lines examined (p-value &amp;lt; 0.01). AR inhibition with Sevi significantly radiosensitized AR+ TNBC xenografts in mouse models and markedly delayed tumor-volume tripling time (TTT) and tumor growth (MDA-MB-453: median TTT 16.1 days for RT alone vs. not reached after 45 days for Sevi+RT, p-value &amp;lt;0.001). Similar delays were seen in tumor growth, weight, and tumor doubling. Clinically, TNBC patients whose tumors had higher than median expression of AR had higher rates of LR after RT (HR for LR ˜3, p-value &amp;lt;0.01, 2 independent datasets). In MVA, high AR expression was the variable most significantly associated with worse LR survival after RT in TNBC patients, outperforming all other variables (HR of 3.42; p-value &amp;lt; 0.01). Conclusions: Our results implicate the AR as a mediator of radioresistance in breast cancer and support the rationale for developing Sevi as a novel radiosensitizing agent in AR+ TNBC. Citation Format: Speers CW, Chandler B, Olsen E, Wilder-Romans K, Moubadder L, Nyati S, Rae J, Hayes DF, Spratt DE, Wahl DR, Eisner J, Feng FY, Pierce LJ. Radiosensitization of androgen receptor (AR)-positive triple-negative breast cancer (TNBC) cells using seviteronel (INO-464), a selective CYP17 lyase and AR inhibitor [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-05.

Cell-line dependent effects of hypoxia prior to irradiation in squamous cell carcinoma lines

PurposeTo assess the impact of hypoxia exposure on cellular radiation sensitivity and survival of tumor cells with diverse intrinsic radiation sensitivity under normoxic conditions. Materials and methodsThree squamous cell carcinoma (SCC) cell lines, with pronounced differences in radiation sensitivity, were exposed to hypoxia prior, during or post irradiation. Cells were seeded in parallel for colony formation assay (CFA) and stained for γH2AX foci or processed for western blot analysis. ResultsHypoxia during irradiation led to increased cellular survival and reduced amount of residual γH2AX foci in all the cell lines with similar oxygen enhancement ratios (OER SKX: 2.31, FaDu: 2.44, UT-SCC5: 2.32), while post-irradiation hypoxia did not alter CFA nor residual γH2AX foci. Interestingly, prolonged exposure to hypoxia prior to irradiation resulted in differential outcome, assessed as Hypoxia modifying factor (HMF) namely radiosensitization (SKX HMF: 0.76), radioresistance (FaDu HMF: 1.54) and no effect (UT SCC-5 HMF: 1.1). Notably, radiosensitization was observed in the ATM-deficient SKX cell line while UT SCC-5 and to a lesser extent also FaDu cells showed radiation- and hypoxia-induced upregulation of ATM phosphorylation. Across all the cell lines Rad51 was downregulated whereas phosphor-DNA-PKcs was enhanced under hypoxia for FaDu and UTSCC-5 and was delayed in the SKX cell line. ConclusionWe herein report a key role of ATM in the cellular fitness of cells exposed to prolonged moderate hypoxia prior to irradiation. While DNA damage response post-irradiation seem to be mainly driven by non-homologous end joining repair pathway in these conditions, our data suggest an important role for ATM kinase in hypoxia-driven modification of radiation response.

Abstract 5839: Androgen receptor (AR): A novel target for radiosensitization in triple-negative breast cancers (TNBC)

Abstract Purpose: Increased rates of local recurrence (LR) have been observed in TNBC despite chemotherapy and radiation (RT). Thus, approaches that result in radiosensitization in TNBC are critically needed. We characterized the RT response of 21 breast cancer cell (BCC) lines using clonogenic survival assays and paired this with high-throughput drug screen data, identifying AR as a top target for radiosensitization. We demonstrate that AR inhibition confers radiosensitization in vitro and in vivo, identified a biomarker of response, and characterize the mechanism of AR-mediated radiosensitization in TNBC. Materials/Methods: Clonogenic survival assays determined the intrinsic RT sensitivity of 21 BCC lines. IC50 values were determined for 130 clinical compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Gene and protein expression was measured using RNA Seq and RPPA arrays, respectively, in tumor samples (n=2,061) and BCC lines (n=51). AR function was assessed using gene knockdown (KD) or functional inhibition with anti-androgen drugs. We measured in vivo tumor growth with varying control and treatment groups (16-20 tumors/group). Kaplan-Meier analysis was performed to estimate local control. A Cox proportional hazards model and MVA were used to determine variables associated with LRF survival. Results: Our unbiased drug radiosensitizer screen nominated bicalutamide as an effective drug in treating RT-resistant BCC lines (R2= 0.46, p-value &amp;lt;0.01). We interrogated the expression of AR in &amp;gt;2000 human breast tumor samples and 51 BCC lines and found heterogeneity in AR expression with strongly correlated expression of protein and RNA levels in TNBC (R2=0.89, p-value &amp;lt;0.001). Inhibition of AR, using both KD and drug (MDV3100) induced RT sensitivity with an enhancement ratio (ER) of 1.35-1.42 in AR+ TNBC lines with no effect on controls. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNAPKcs. AR inhibition with MDV3100 significantly radiosensitized TNBC xenografts in mouse models and markedly delayed tumor tripling time (TTT) and tumor growth (median TTT 17.4 days for RT alone vs. not reached after 50 days for MDV3100+RT, p-value &amp;lt;0.001). Activated DNAPK was identified as a biomarker of response. Clinically, TNBC patients whose tumors had higher than median expression of AR had higher rates of LR after RT (HR for LR ~3, p-value &amp;lt;0.01, 2 independent datasets). In MVA, high AR expression was the variable most significantly associated with worse LRF survival after RT in TNBC patients, outperforming all other variables (HR of 3.58; p-value &amp;lt; 0.01). Conclusion: Our results implicate AR as a mediator of radioresistance in breast cancer and support the rationale for developing clinical strategies, including clinical trials, to inhibit AR as a novel radiosensitizing target in TNBC. Citation Format: Benjamin C. Chandler, Corey W. Speers, Shuang G. Zhao, Meilan Liu, Kari Wilder-Romans, Eric Olsen, Shyam Nyati, Daniel Spratt, Daniel Wahl, Daniel Hayes, Felix Y. Feng, Lori J. Pierce. Androgen receptor (AR): A novel target for radiosensitization in triple-negative breast cancers (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5839. doi:10.1158/1538-7445.AM2017-5839

Zerumbone Regulates DNA Repair Responding to Ionizing Radiation and Enhances Radiosensitivity of Human Prostatic Cancer Cells.

Introduction. Radiation therapy using ionizing radiation is widely used for the treatment of prostate cancer. The intrinsic radiation sensitivity of cancer cells could be enhanced by modulating multiple factors including the capacity to repair DNA damage, especially double-strand breaks (DSBs). We aimed to examine the effect of zerumbone on radiation sensitivity and its protective effects against ionizing radiation–induced DSB in human prostate cancer cells. Materials and Methods. The human prostate cancer PC3 and DU145 cell lines were used. A colony formation assay was performed to analyze the radiation survival of cells. DNA histogram and generation of reactive oxygen species (ROS) were examined using flow cytometry. Western blotting was used to examine the expression of regulatory molecules related to DNA damage repair. Results. Pretreatment with zerumbone enhanced the radiation effect on prostate cancer cells. Zerumbone delayed the abrogation of radiation-induced expression of γ-H2AX, an indicator of DNA DSB. Zerumbone pretreatment markedly reduced ionizing radiation–induced upregulated expression of phosphorylated ATM (ataxia telangiectasia-mutated), which was partially reversed by the ATM agonist methyl methanesulfonate. Ionizing radiation augmented and zerumbone pretreatment reduced the expression of Jak2 and Stat3, which are involved in DNA damage repair signaling. No significant effect on the generation of ROS and expression of ATR was noted after zerumbone treatment. Conclusion: Zerumbone sensitized DU145 and PC3 prostatic cancer cells to ionizing radiation by modulating radiation-induced ATM activation during repair of DNA DSBs.

Radiosensitization of androgen receptor (AR)-positive triple-negative breast cancer (TNBC) cells using seviteronel (SEVI), a selective CYP17 lyase and AR inhibitor.

e12102 Background: Increased rates of locoregional recurrence have been observed in TNBC despite chemotherapy and radiation (RT). A novel radiosensitizer screen nominated the AR as a promising target for treatment of radioresistant breast cancer, including TNBC. We assessed in vitro activity of SEVI (VT-464), a selective CYP17 lyase and AR inhibitor, as a potential radiosensitizer in AR+ TNBC model. Methods: Clonogenic survival assays were used to determine the intrinsic RT sensitivity of 21 breast cancer cell (BCC) lines. IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Gene expression was measured using RNA Seq or qRT-PCR and protein expression was measured using RPPA arrays. AR function was assessed using functional inhibition with SEVI in MDA-MB-453, ACC-460, SUM-185 (all three AR+ TNBC), MDA-MB-231 (AR- TNBC), and T47D (AR- ER+) BCC lines. Double-stranded DNA (dsDNA) break repair was assessed with γH2AX foci counting. Results: Our novel radiosensitizer screen identified the activity of bicalutamide, an AR antagonist, in RT-resistant BCC lines (R2 = 0.46, p-value &lt; 0.01). Heterogeneity in AR expression was identified in human BCC lines. There was a strong correlation between AR RNA expression and protein expression across all BC intrinsic subtypes. AR inhibition using SEVI induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.24-1.62 in three different AR+ TNBC lines. No such radiosensitization was seen in AR(-) TNBC or ER+, AR(-) BCC lines. Radiosensitization was at least partially dependent on impaired dsDNA break repair with significant delays in dsDNA break repair at 16 and 24 hours in all AR+ TNBC lines examined (p-value &lt; 0.01). Conclusions: Our results implicate the AR as a mediator of radioresistance in breast cancer and support the rationale for developing seviteronel as a novel radiosensitizing agent in AR+ TNBC.

815 - Ex vivo γH2AX assay in prostate cancer patient-derived tumour samples reveals substantial differences in intrinsic radiation sensitivity

815 - Ex vivo γH2AX assay in prostate cancer patient-derived tumour samples reveals substantial differences in intrinsic radiation sensitivity

Enhanced binding of necrosis-targeting immunocytokine NHS-IL12 after local tumour irradiation in murine xenograft models.

NHS-IL12 is an immunocytokine targeting necrotic tumour areas. IL12 shows anti-tumour activity. As local irradiation might induce additional necrosis in solid tumours, we aimed to evaluate the increase in intratumoural accumulation of NHS-IL12 after irradiation and correlate the findings with diffusion-weighted MRI studies in two xenograft models. Human rhabdomyosarcoma (A204) and prostate cancer (PC3) cells were studied in vitro and as subcutaneous xenografts. Radiation sensitivity of the cell lines was assessed in vitro by colony formation assays. In vivo tumour necrosis was assessed based on apparent diffusion coefficients (ADC). Biodistribution of NHS-IL12 was evaluated with and without tumour irradiation using in vivo small-animal PET and ex vivo biodistribution. A204 and PC3 differed in their intrinsic radiation sensitivity. Accordingly, radiation-induced tumour necrosis was found only in A204 xenografts. In comparison with control, ADC was significantly increased after irradiation of A204 tumours with 1×8.0Gy and 5×2.0Gy, whereas no change in ADC was observed in PC3 xenografts in all irradiation regimes. ADC correlated with histology. An enhanced uptake of radiolabelled NHS-IL12 in A204 tumours was detected by PET and ex vivo biodistribution after tumour irradiation. In PC3 tumours, no increase in NHS-IL12 uptake was observed. In dependence of the tumour model, tumour irradiation enhanced tumour necrosis measured in MRI and histology. In vivo PET and ex vivo biodistribution showed enhanced binding of NHS-IL12 in rhabdomyosarcoma xenografts. Thus, enhanced binding of necrosis-targeting immunocytokines might be a novel mechanism of additive effects in combination with irradiation.

Abstract P6-13-05: Androgen receptor (AR): A novel target and mechanism for radiosensitization and treatment in triple-negative breast cancers (TNBC)

Abstract Background: Increased rates of locoregional recurrence have been observed in TNBC despite chemotherapy and radiation (RT). Thus, approaches that result in radiosensitizaton in TNBC are critically needed. We characterized the RT response of 21 breast cancer cell (BCC) lines using clonogenic survival assays. We paired this with high-throughput drug screen data to identify AR as a top target for radiosensitization and assess AR inhibition as a radiosensitization strategy for TNBC. Methods: Clonogenic survival assays were used to determine the intrinsic RT sensitivity of 21 BCC lines. IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values and SF-2Gy. Gene expression was measured using RNA Seq and protein expression was measured using RPPA arrays in human tumor samples (n=2,061) and BCC lines (n=51). AR function was assessed using siRNA knockdown or functional inhibition with MDV3100 (enzalutamide). We measured in vivo tumor growth with varying control and treatment groups (16-20 tumors/group). Kaplan-Meier analysis was performed to estimate local control and survival. A Cox proportional hazards model was used to identify factors of survival, and MVA was used to determine variables associated with LRF survival. Results: Our radiosensitizer screen nominated bicalutamide as a most effective drug in treating RT-resistant BCC lines (R2= 0.46, p-value &amp;lt;0.01). We interrogated the expression of AR in &amp;gt;2000 human breast tumor samples and found significant heterogeneity in AR expression with enrichment of expression at the protein and RNA level in TNBC. This same heterogeneity was also identified in human BCC lines. There was a strong correlation between AR RNA expression and protein expression (R2= 0.72, p &amp;lt;0.01). Inhibition of AR using both siRNA and MDV3100 (enzalutamide) induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.35-1.42 in AR-positive TNBC lines. No such radiosensitization was seen in AR-negative TNBC or ER-positive, AR-negative BCC lines. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNAPKcs. AR inhibition either with MDV3100 significantly radiosensitized TNBC xenografts in mouse models and markedly delayed tumor tripling time and tumor growth (median tumor tripling time 17.4 days for RT alone vs. not reached after 50 days for MDV3100 + RT, p-value &amp;lt;0.001). Biomarker analysis identifies DNAPKcs as a potential biomarker of response. Clinically, analyses of patients with TNBC showed that patients whose tumors had higher than median expression of AR had markedly higher rates of LR after RT (HR for LR 2.9-3.2, p-value &amp;lt;0.01, 2 independent datasets). There was no difference in LR in TNBC patients not treated with RT when stratified by AR expression. In MVA, AR expression was the variable most significantly associated with worse LRF survival after RT (HR of 3.58;p-value &amp;lt; 0.01). Conclusion: Our results implicate AR as a mediator of radioresistance in breast cancer and support the rationale for developing clinical strategies, including clinical trials, to inhibit AR as a novel radiosensitizing target in TNBC. Citation Format: Speers C, Zhao SG, Liu M, Rae JM, Hayes DF, Feng FY, Pierce LJ. Androgen receptor (AR): A novel target and mechanism for radiosensitization and treatment in triple-negative breast cancers (TNBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-05.

Residual γH2AX foci after ex vivo irradiation of patient samples with known tumour-type specific differences in radio-responsiveness

PurposeTo apply our previously published residual ex vivo γH2AX foci method to patient-derived tumour specimens covering a spectrum of tumour-types with known differences in radiation response. In addition, the data were used to simulate different experimental scenarios to simplify the method. Materials and methodsEvaluation of residual γH2AX foci in well-oxygenated tumour areas of ex vivo irradiated patient-derived tumour specimens with graded single doses was performed. Immediately after surgical resection, the samples were cultivated for 24h in culture medium prior to irradiation and fixed 24h post-irradiation for γH2AX foci evaluation. Specimens from a total of 25 patients (including 7 previously published) with 10 different tumour types were included. ResultsLinear dose response of residual γH2AX foci was observed in all specimens with highly variable slopes among different tumour types ranging from 0.69 (95% CI: 1.14–0.24) to 3.26 (95% CI: 4.13–2.62) for chondrosarcomas (radioresistant) and classical seminomas (radiosensitive) respectively. Simulations suggest that omitting dose levels might simplify the assay without compromising robustness. ConclusionHere we confirm clinical feasibility of the assay. The slopes of the residual foci number are well in line with the expected differences in radio-responsiveness of different tumour types implying that intrinsic radiation sensitivity contributes to tumour radiation response. Thus, this assay has a promising potential for individualized radiation therapy and prospective validation is warranted.

High dose rate and flattening filter free irradiation can be safely implemented in clinical practice

Purpose: We hypothesize that flattening filter free (FFF) high dose rate irradiation will decrease cell survival in normal and cancer cells with more pronounced effects in DNA repair deficient cells. Additionally, we hypothesize that removal of the flattening filter will result in an enhanced relative biological effectiveness independent of the dose rate.Materials and methods: Clonogenic survival was assessed after exposure to dose rates of 4 or 24 Gy/min (FFF 10 megavolt [MV] photon beam) using a Varian TrueBeam accelerator. Additionally, cells were exposed to 4 Gy/min with or without flattening filter. Relative biological effectiveness estimations were performed comparing the different beam photon spectra.Results: Cell survival in tumor and normal cell lines was not influenced by high dose rate irradiation. The intrinsic radiation sensitivity of DNA repair deficient cells was not affected by high dose rate compared to normal dose rate. Furthermore, the relative biological effectiveness was not significantly different from unity in any of the cell lines for both FFF and conventional flattened beam exposures.Conclusions: High dose rate irradiation did not affect long-term survival and DNA repair for cell lines of different tissues. This suggests that high dose rate does not influence treatment outcome or treatment toxicity and could be safely implemented in clinical routine.

The radiosensitizing activity of the SMAC-mimetic, Debio 1143, is TNFα-mediated in head and neck squamous cell carcinoma

Background and purposeSecond mitochondria-derived activator of caspase (SMAC)-mimetics are a new class of targeted drugs that specifically induce apoptotic cancer cell death and block pro-survival signaling by antagonizing selected members of the inhibitor of apoptosis protein (IAP) family. Material and methodsThe present study was designed to investigate the radiosensitizing effect and optimal sequence of administration of the novel SMAC-mimetic Debio 1143 in vitro and in vivo. Apoptosis, alteration of DNA damage repair (DDR), and tumor necrosis factor-alpha (TNF-α) signaling were examined. ResultsIn vitro, Debio 1143 displayed anti-proliferative activity and enhanced intrinsic radiation sensitivity in 5/6 head and neck squamous cell carcinoma (HNSCC) cell lines in a synergistic manner. In vivo, Debio 1143 dose-dependently radio-sensitized FaDu and SQ20B xenografts, resulting in complete tumor regression in 8/10 FaDu-xenografted mice at the high dose level. At the molecular level, Debio 1143 combined with radiotherapy (RT) induced enhancement of caspase-3 activity, increase in Annexin V-positive cells and karyopyknosis, and increase in TNF-α mRNA levels. Finally, in a neutralization experiment using a TNF-α-blocking antibody and a caspase inhibitor, it was shown that the radiosensitizing effect of Debio 1143 is mediated by caspases and TNF-α. ConclusionsThese results demonstrate that the novel SMAC-mimetic Debio 1143 is a radiosensitizing agent that is worthy of further investigation in clinical trials in combination with radiotherapy.

Abstract P6-03-08: Androgen receptor (AR): A novel target for radiosensitization and treatment in triple-negative breast cancers (TNBC)

Abstract Background: Increased rates of locoregional recurrence have been observed in TNBC despite the use of chemotherapy and radiation (RT). Thus, approaches that result in radiosensitizaton of TNBC are critically needed. We have previously characterized the radiation response of 21 breast cancer cell (BCC) lines using clonogenic survival assays. We now pair this data with high-throughput drug screen data available through cancer cell line encyclopedia studies to identify AR as a top target for radiosensitization and assess AR inhibition as a radiosensitization strategy for TNBC. Methods: Clonogenic survival assays were performed to determine the intrinsic RT sensitivity of 21 BCC lines (0-8 Gy RT). IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values (for drug sensitivity) and SF-2Gy (for radiation sensitivity). Gene expression was measured using Affymetrix microarrays and protein expression was measured using reverse-phase protein lysate arrays (RPPA) of human tumor samples (n=2,061) and BCC lines (n=51). AR function was assessed using siRNA knockdown or inhibition with MDV3100 (enzalutamide). Kaplan-Meier analysis was performed to determine the clinical impact of AR expression on local control and survival. A Cox proportional hazards model was constructed to identify potential factors of survival, and multivariate analysis was used to determine variables most significantly associated with LRF survival. Results: Our radiosensitizer screen nominated bicalutamide as one of the most effective drugs in treating radioresistant BCC lines (R2= 0.46, p-value &amp;lt;0.001). Recognizing that a subgroup of TNBC includes AR expressing tumors, we interrogated the expression of AR in &amp;gt;2000 human breast tumor samples and found signifi[not]cant heterogeneity in AR expression with an increase in TNBC (35% of tumors) compared to non-TNBC (28% of tumors). This same heterogeneity was also identified in human BCC lines. There was a strong correlation between AR RNA expression and protein expression (R2= 0.72, p &amp;lt;0.0001). Inhibition of AR using both siRNA and MDV3100 induced radiation sensitivity in vitro with an enhancement ratio (ER) of 1.35-1.42 in AR-positive TNBC lines. No such radiosensitization was seen in AR-negative TNBC or ER-positive, AR-negative BCC lines. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNAPKcs. In vivo assessment of tumor growth inhibition with RT and anti-AR strategies are currently underway. Clinically, analyses of patients with TNBC showed that patients whose tumors had high expression of AR had markedly higher rates of LR after RT than patients with low expression of AR (HR for LR 2.9-3.2, p-value &amp;lt;0.01, 2 independent datasets). There was no difference in LR in TNBC patients not treated with RT when stratified by AR expression status. In multivariate analysis, AR expression was the variable most significantly associated with worse LRF survival after RT with a HR of 3.58 (p-value &amp;lt; 0.01). Conclusion: Our results implicate AR as a mediator of radioresistance in breast cancer and support the rationale for developing clinical strategies to inhibit AR as a novel radiosensitizing target in TNBC. Citation Format: Corey Speers, Shuang G Zhao, Meilan Liu, Joseph Evans, Prasanna Alluri, Daniel F Hayes, Felix Y Feng, Lori J Pierce. Androgen receptor (AR): A novel target for radiosensitization and treatment in triple-negative breast cancers (TNBC) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-08.