Radiation Response Of Tumors Research Articles

Targeting Cancer Metabolism to Enhance Tumor Radiation Response

A key emerging hallmark of cancer includes a deregulated cellular metabolism that can be reprogrammed to preferentially exhibit dependence on glycolysis over oxidative phosphorylation (OXPHOS) even in the presence of oxygen (Warburg effect). Pre-clinical data suggests that targeting lactate dehydrogenase (LDH) enzyme and components of mitochondrial OXPHOS metabolism in tumor cells can have a potent anti-tumor effects, yet the interactions of these anti-tumor approaches with standard therapies, such as RT is less clear. clonogenic assay, immunoblotting, tumor regrowth delay, Seahorse studies, LC/MS Mass spectrometry We characterized novel and specific inhibitors targeting LDH enzyme A and B isoform (LDHi) and novel OXPHOS inhibitor (IACS-010759) targeting mitochondrial complex I in combination with ionizing radiation (IR) for their anti-cancer and radio-sensitization effects across various tumor types. Targeting LDH enzyme in vitro leads to significant (p<0.001) reduction in extracellular lactate levels and radiosensitization of variety of tumor cell lines with a dose modification factor (DMF) ranging between 1.5-2.2. Clonogenic survival studies of variety of pancreatic, prostate and lung cancer cells indicated that inhibiting LDH in conjunction with IR, can also enhance radiosensitivity under both hypoxic (1% oxygen) (DMF = 2.0) and normoxic (DMF = 1.65) conditions while not affecting non-glycolytic/normal cells (1522, skin fibroblasts, DMF = 1.0) in vitro. Further, we established that this enhanced radiosensitivity upon LDH inhibition could be partly attributed to reduced DNA repair as seen by enhanced expression of gamma-H2AX (4 fold), reduced ATP generation (80% reduction compared to control) as well as altered expression of glucose and lactate transporter proteins (Glut-1, MCT-1, MCT-4, PFKP and PKM2) as seen by immunoblotting. Tumor regrowth delay studies with MiaPaCa and H460 xenografts demonstrated differential LDH response with no significant tumor radiosensitization. We attributed this failure to tumor metabolic reprogramming confirmed by increased oxygen consumption rate of tumor cells upon LDH inhibition in vitro. Targeting OXPHOS pathway by IACS-010759 in conjunction with LDHi promoted metabolic synthetic lethality in vitro (survival reduced to 10-5). Similar synthetic lethality was also observed in the H460 xenograft; additive enhancement in radiation response. However, inhibition of OXPHOS by complex I inhibitor and fractionated IR led to additive tumor regrowth delay in both MiaPaCa and H460 xenograft models. Furthermore, steady state global metabolomics study of inhibiting LDH and complex I of mitochondria either alone or in combination lead to significant decrease in glycolytic and TCA cycle intermediates leading to severe energy stress. In summary, our results presently indicate that targeting mitochondrial metabolism may be a more effective means of enhancing tumor radiosensitivity.

Abstract 6284: GC4419 protects again radiation induced oral mucositis, enhances the response of squamous cell carcinoma of the head and neck tumors to ionizing radiation, and enhances radioimmune therapy

Abstract Locoregional recurrence (LRR) is the major cause of morbidity and mortality in patients with squamous cell carcinoma of the head and neck (HNSCC). For those patients that experience surgically unresectable LRR, median overall survival (OS) is less than one year. Approaches utilizing stereotactic ablative radiotherapy (SAbR) and immune oncology agents such as Nivolumab (α-PD-1 agonist) in the salvage setting have become alternatives to conventional chemoradiation therapy. Despite improvements in OS, rates of acute oral mucositis (OM) and late toxicities (skin fibrosis) remain a concern with SAbR, particularly in the context of re-irradiation. Here we present pre-clinical evidence that GC4419 (Galera Therapeutics), a highly selective superoxide dismutase mimetic that recently received FDA Breakthrough therapy status following a Phase II, randomized, placebo controlled trial for reducing the duration and incidence of severe OM in patients undergoing chemoradiation therapy for locally advanced HNSCC (NCT02508389) also enhances the anti-tumor radiation response of HNSCC tumors to irradiation. We demonstrate in pre-clinical animal models that GC4419 protects the normal mucosa of the mouse tongue from radiation exposure. Utilizing clonogenic survival assays, GC4419 demonstrates single agent anti-cancer activity at physiologically achievable concentrations, while also enhancing the response of HNSCC lines to irradiation. In tumor growth delay (TGD) experiments utilizing the syngeneic HNSCC tumor model, AT-84, when GC4419 is delivered 30-60 minutes pre-irradiation with biologically equivalent fractionation schedules of 17 Gy x 1 fxn, 10.24 Gy x 2 fxn, or 5 Gy x 5 fxn, an enhancement of the radiation response is achieved. In tumor cure rate (TCD50) studies, GC4419 enhanced radiation exposure with a DEF of 1.25. Furthermore, when GC4419 is combined with radiation and an α-PD-1 inhibitor, a further enhancement of the radiation response is observed, indicating that GC4419 compliments radiotherapy combined with immune oncology. We will also report the results of ongoing studies examining the potency of GC4419 in protecting normal murine mucosa in the setting of reirradiation relevant to SAbR as a salvage therapy. This potentiation of the anti-tumor response with GC4419 is more pronounced at doses exceeding the threshold to be considered for intensity modulated radiation therapy (IMRT). The accompanying pre-clinical data strongly suggest that GC4419 should be combined with radio-immune therapy to not only enhance local tumor control, but that the potential for normal tissue protection with SAbR also creates the opportunity for dose escalation and may further improvement in treatment outcome. Citation Format: Brock James Sishc, Elizabeth Polsdofer, Debabrata Saha, Michael Story. GC4419 protects again radiation induced oral mucositis, enhances the response of squamous cell carcinoma of the head and neck tumors to ionizing radiation, and enhances radioimmune therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6284.

Angiotensin II type 1 receptor blocker telmisartan inhibits the development of transient hypoxia and improves tumour response to radiation.

Hypoxic tumour cells are radiation-resistant and are associated with poor therapeutic outcome. A poorly understood source of tumour hypoxia is unstable perfusion, which exposes tumour cells to varying oxygen tensions over time creating "transiently" hypoxic cells. Evidence suggests that angiotensin II type 1 receptor blockers (ARBs) can improve tumour perfusion by reducing collagen deposition from cancer associated fibroblasts (CAFs). However, the influence of ARBs on transient hypoxia and tumour radiation response is unknown. We tested how the ARBs losartan and telmisartan affected the solid tumour microenvironment, using fluorescent perfusion dyes and positron emission tomography to quantify tumour perfusion, and a combination of hypoxia markers and the hemorheological agent pentoxifylline to assess transient tumour hypoxia. We found CAF-containing tumours have reduced collagen I levels in response to telmisartan, but not losartan. Telmisartan significantly increased tumour blood flow, stabilized microregional tumour perfusion, and decreased tumour hypoxia by reducing the development of transient hypoxia. Telmisartan-treated tumours were more responsive to radiation, indicating that telmisartan reduces a therapeutically important population of transiently hypoxic tumour cells. Our findings indicate telmisartan is capable of modifying the tumour microenvironment to stabilize tumour perfusion, reduce transient hypoxia, and improve tumour radiation response.

TGF\u03b2 suppresses CD8+ T cell expression of CXCR3 and tumor trafficking

Transforming growth factor beta (TGFβ) is a multipotent immunosuppressive cytokine. TGFβ excludes immune cells from tumors, and TGFβ inhibition improves the efficacy of cytotoxic and immune therapies. Using preclinical colorectal cancer models in cell type-conditional TGFβ receptor I (ALK5) knockout mice, we interrogate this mechanism. Tumor growth delay and radiation response are unchanged in animals with Treg or macrophage-specific ALK5 deletion. However, CD8αCre-ALK5flox/flox (ALK5ΔCD8) mice reject tumors in high proportions, dependent on CD8+ T cells. ALK5ΔCD8 mice have more tumor-infiltrating effector CD8+ T cells, with more cytotoxic capacity. ALK5-deficient CD8+ T cells exhibit increased CXCR3 expression and enhanced migration towards CXCL10. TGFβ reduces CXCR3 expression, and increases binding of Smad2 to the CXCR3 promoter. In vivo CXCR3 blockade partially abrogates the survival advantage of an ALK5ΔCD8 host. These data demonstrate a mechanism of TGFβ immunosuppression through inhibition of CXCR3 in CD8+ T cells, thereby limiting their trafficking into tumors.

Усиленная радиосенсибилизация опухолевых клеток с помощью сочетанного воздействия гипертермии и ингибиторов протеасом

Hyperthermia is used in combination with radiation therapy to enhance the radiation response of the target tumor. However, many tumors are thermoresistant, which makes the thermo-radiosensitization ineffective. In this work, we studied a possibility of enhancing the radiosensitiz-ing effect of hyperthermia on cancer cells in the case of combining heating and treatments with inhibitors of proteasomal protein degradation. The objects of the study were HeLa, MCF-7, and PC-3 cell lines derived from human carcinomas of various localization. Before irradiation (2-6 Gy), cell cultures were subjected to heat stress (42-44 °C for 20-60 min) without or in the pres-ence of proteasome inhibitors (MG132, bortezomib). The death and survival of the treated cells was evaluated by the level of apoptosis/necrosis, by clonogenicity, and in the MTT test. The pro-teotoxicity of the exposures and the subcellular redistribution of heat shock proteins were ana-lyzed using cell fractionation, electrophoresis, and immunoblotting. The results show that, com-pared with hyperthermia, the combined effect (hyperthermia + proteasome inhibitor) significantly increased the radiosensitization of cancer cells, which was manifested in the intensification of their postradiation death and significant suppression of clonogenicity. Thus, the combination of hyperthermia with proteasome inhibitors (for example, with bortezomib, which is clinically ap-proved) can help sensitize radioresistant tumors to radiation therapy.

Role of Bee Venom and Melittin on Restraining Angiogenesis and Metastasis in γ-Irradiated Solid Ehrlich Carcinoma-Bearing Mice.

Pathological angiogenesis and apoptosis evasion are common hallmarks of cancer. The present work was an endeavor to evaluate the influence of bee venom (BV) or its major constituent melittin (MEL) as antiapoptotic and angiogenic regulator modifier on the tumor growth and the cell sensitivity to ionizing radiation targeting the improvement of cancer therapeutic protocols. BV (0.56 mg/kg/day) and MEL (500 µg/kg body weight/day) were injected intraperitoneally to mice bearing 1 cm3 solid tumor of Ehrlich ascites carcinoma (EAC) for 21 consecutive days. Mice were whole-body exposed to 1 Gray (Gy) of γ-radiation (2 fractionated doses). Treatment with BV or MEL markedly suppresses the proliferation of tumor in EAC mice. The concentrations of m-RNA for angiogenic factors (TNF-α, VEGF) as well as MMPs 2 and 9 activities and NO concentration were significantly decreased, combined with improvements in apoptotic regulators (caspase-3 activity) and normal cells redox tone (catalase and free radicals content) compared with EAC mice. Moreover, the histopathological investigation confirms the improvement exerted by BV or MEL in the EAC mice group or EAC + R group. Exposure to γ-radiation sustained the modulatory effect of BV on tumor when compared with EAC + BV mice. Convincingly, the role of BV or MEL as a natural antiangiogenic in the biological sequelae after radiation exposure is verified. Hence, BV and its major constituent MEL might represent a potential therapeutic strategy for increasing the radiation response of solid tumors.

Harnessing Tumor Immune Ecosystem Dynamics to Personalize Radiation Therapy

Radiotherapy is a pillar of cancer care and augments the response to immunotherapies. However, little is known regarding the relationships between the tumor immune ecosystem (TIES) and intrinsic radiosensitivity. A pressing question in oncology is how to optimize radiotherapy to improve patient responses to immune therapies. To address this, we profiled 10,469 primary tumors for metrics of radiosensitivity and immune cell infiltrate (ICI), and applied a new integrated in silico model that mimics the dynamic relationships between tumor growth, ICI flux and radiation response. We validated this model in 10,469 primary tumors and a separate cohort of 59 lung cancer patients treated with radiotherapy. These analyses explain radiation response based on its effect on the TIES and quantifies the likelihood that radiation can promote a shift to anti-tumor immunity. Dynamic modeling of the relationship between tumor radiosensitivity and the TIES may provide opportunity to personalize combined radiation and immunotherapy approaches.

System biological and experimental validation of miRNAs target genes involved in colorectal cancer radiation response

Background and objectivesPrediction of response to radiotherapy in colorectal cancer (CRC) patients is of great interest that protects radiation-resistant patients from imposing to high doses of X radiation. miRNAs and their target genes are potential candidates as biomarkers of cancer. Microarray provides a high-throughput analysis of cell transcriptome; however, this very complex data needs to be interpreted via network-based approaches. In this study, miRNAs target genes involved in the CRC radiation response were validated through a system biological and experimental manner. Material and methodsThe microarray expression data was obtained from NCBI GEO datasets. The miRNAs target genes were identified using miRNA-mRNA interaction prediction databases. Gene enrichment analysis performed using DAVID database to identify genes involved in the tumor radiation response. For selected miRNAs and targets, the miRNA-regulated protein–protein interactions network (PPIN) was constructed and analyzed using Cytoscape software. Survival analysis was done by Xena Browse. Radioresistant cell lines were established using fractional X irradiations and confirmed by clonogenic assay. After RNA isolation and cDNA synthesis, by Real-Time PCR, gene expression measurements were performed. SPSS V.22 was used for data analysis. ResultsFinally, the selected genes and miRNAs based on network analysis were comprises: mTOR, MAPK1, CDC42, EGFR, MAPK8, CCND1, BCL2, PIK3CG, hsa-miR-7-2, hsa-miR-17, hsa-miR-106a and hsa-miR-32. Among them, MAPK8 and CDC42 genes were significantly correlated with CRC patient's survival. Established radioresistant cell lines showed a morphological change and a significantly higher survival fraction compared to parental cell line. Real-Time PCR showed higher expression of MAPK8, CDC42 and CCND1 in radioresistant cell lines compared to parental cell line. ConclusionIn this study, via several enrichment stages, 2,069,706 target genes were highly enriched to 3 genes which have crucial roles in tumor radiation response and can be used as potential predictive biomarkers.

The CD73/Ado System-A New Player in RT Induced Adverse Late Effects.

Radiotherapy (RT) is a central component of standard treatment for many cancer patients. RT alone or in multimodal treatment strategies has a documented contribution to enhanced local control and overall survival of cancer patients, and cancer cure. Clinical RT aims at maximizing tumor control, while minimizing the risk for RT-induced adverse late effects. However, acute and late toxicities of IR in normal tissues are still important biological barriers to successful RT: While curative RT may not be tolerable, sub-optimal tolerable RT doses will lead to fatal outcomes by local recurrence or metastatic disease, even when accepting adverse normal tissue effects that decrease the quality of life of irradiated cancer patients. Technical improvements in treatment planning and the increasing use of particle therapy have allowed for a more accurate delivery of IR to the tumor volume and have thereby helped to improve the safety profile of RT for many solid tumors. With these technical and physical strategies reaching their natural limits, current research for improving the therapeutic gain of RT focuses on innovative biological concepts that either selectively limit the adverse effects of RT in normal tissues without protecting the tumor or specifically increase the radiosensitivity of the tumor tissue without enhancing the risk of normal tissue complications. The biology-based optimization of RT requires the identification of biological factors that are linked to differential radiosensitivity of normal or tumor tissues, and are amenable to therapeutic targeting. Extracellular adenosine is an endogenous mediator critical to the maintenance of homeostasis in various tissues. Adenosine is either released from stressed or injured cells or generated from extracellular adenine nucleotides by the concerted action of the ectoenzymes ectoapyrase (CD39) and 5′ ectonucleotidase (NT5E, CD73) that catabolize ATP to adenosine. Recent work revealed a role of the immunoregulatory CD73/adenosine system in radiation-induced fibrotic disease in normal tissues suggesting a potential use as novel therapeutic target for normal tissue protection. The present review summarizes relevant findings on the pathologic roles of CD73 and adenosine in radiation-induced fibrosis in different organs (lung, skin, gut, and kidney) that have been obtained in preclinical models and proposes a refined model of radiation-induced normal tissue toxicity including the disease-promoting effects of radiation-induced activation of CD73/adenosine signaling in the irradiated tissue environment. However, expression and activity of the CD73/adenosine system in the tumor environment has also been linked to increased tumor growth and tumor immune escape, at least in preclinical models. Therefore, we will discuss the use of pharmacologic inhibition of CD73/adenosine-signaling as a promising strategy for improving the therapeutic gain of RT by targeting both, malignant tumor growth and adverse late effects of RT with a focus on fibrotic disease. The consideration of the therapeutic window is particularly important in view of the increasing use of RT in combination with various molecularly targeted agents and immunotherapy to enhance the tumor radiation response, as such combinations may result in increased or novel toxicities, as well as the increasing number of cancer survivors.

1954P - Serum exosomal microRNA-199b-5p as a novel circulating biomarker to predict response of preoperative chemoradiotherapy for locally advanced rectal cancer

BackgroundThe effect of preoperative chemoradiotherapy (CRT) varies from complete response to complete resistance, and predicting response to CRT have not been well characterized yet. Previous studies have shown the potential of micro-RNA (miRNA) based approaches to enhance tumor radiation response. Accordingly, the present study attempted to identify biomarkers to predict response for preoperative CRT using comprehensive miRNA analysis in patients with LARC. MethodsThis study included 65 rectal cancer tissues and 90 serum samples from patients who diagnosed with LACR and received preoperative CRT at Kyungpook National University Chilgok Hospital. Tissue specimens and serum samples were collected before CRT to evaluate the biologic differences between the good CRT response group and the poor CRT response group. For discovery of specific miRNAs, 800 miRNAs were analyzed using NanoString in 30 rectal cancer tissues. Thereafter, a total of 65 tissues, and 90 serum samples were investigated using real-time PCR for validation. ResultsThe pathologic stages after preoperative CRT were as follows: pathologic complete response (n=13, 14.4%), pathologic stage I (n=13, 14.4%), pathologic stage II (n=27, 30.0%), pathologic stage III (n=28, 31.1%), and pathologic stage IV (n=9, 10.0%). In the discovery set, 16 target miRNAs were detected. In the validation set with tissue specimens, expression of 3 miRNAs (miR-199a/b-3p (p=0.032), miR-199a-5p (p=0.023), miR-199b-5p (p=0.005)) was significantly upregulated which was associated with better response of CRT. Moreover, among the 3 candidate miRNAs, miR-199b-5p level was significantly upregulated in serum, and it was also found to be related with better response of CRT in LARC (pathologic stage 0/I versus II/III/IV, p=0.027). ConclusionsIn the present study, high level of serum miR-199b-5p was associated with better response, suggesting it to be a promising non-invasive biomarker to predict response of CRT in patients with LARC. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Targeted next-generation DNA sequencing identifies Notch signaling pathway mutation as a predictor of radiation response

Purpose: Identifying the association between somatic mutations and the radiation response of tumor is essential for understanding the mechanisms and practicing personalized radiotherapy. The present study aimed to discover specific genes or pathways that are associated with radiation response using targeted next-generation DNA sequencing.Material and methods: Fifty-five patients with various solid tumors whose specimen were sequenced using institutional panel which includes 148 cancer-related genes and received radiotherapy for a measurable tumor were analyzed. Patients with irradiated tumors in complete or partial remission for more than 6 months were defined as responders. Association between mutations including pathogenic single nucleotide variants and insertions/deletions in the 148 genes and 39 molecular pathways and radiation response was investigated.Results: Analyzing 17 responders and 38 non-responders, biologically effective dose (BED), but not concurrent chemotherapy, was associated with radiation response. No single gene correlated with radiation response. Mutations in Notch signaling pathway were associated with radiosensitivity after correction for multiple comparison (adjusted p = .094). When BED and Notch signaling pathway mutation were tested with logistic regression, both variables were associated with radiation response.Conclusions: Our results suggest that somatic mutations in Notch signaling pathway may be related to sensitivity to radiation, although these results should be validated in a larger and more homogeneous cohort.

Proliferation Saturation Index to Prospectively Predict Patient-Specific Responses to Radiation in Oropharyngeal Cancer

Mid-treatment response to radiation therapy is prognostic for ultimate outcome in patients with head and neck cancer (HNC). For patients predicted to not achieve robust mid-treatment responses (tumor volume reduction less than 32% by week 4 of daily 2 Gy fraction), alternative radiation protocols may be selected. To this extent, a predictive model is needed to forecast individual response to radiation based on pre-treatment data. The Proliferation Saturation Index (PSI) model has demonstrated excellent fits to radiation response data in multiple cancers, including oropharyngeal cancer (OPC). We hypothesize that PSI model parameters can be learned from a training data set, and be applied to pre-treatment radiology images at diagnosis, treatment simulation, or before first radiation fraction to predict patient-specific radiation responses of patients in a test cohort. We use a mathematical model with three parameters including patient-uniform tumor growth and radiation response rates, and patient-specific PSI. Two pre-treatment and one mid-treatment radiology scans were delineated to derive longitudinal tumor volumes of 15 OPC patients. In a leave-one-out approach, the model parameters are learned to maximize predictive power in the training set. The derived patient-uniform parameters are then applied to the patient on which the model was not trained to predict mid-treatment radiation response. Model outcome performance is evaluated based on classification accuracy, including sensitivity and specificity to predict mid-treatment response. Patient-uniform OPC growth rate is determined to be 0.04 day-1, and radiation response rate 0.172 day-1(0.156-0.204). From the trained parameters, average patient-specific PSI is 0.83 +/- 0.25 (0.075-0.99). Mid-treatment radiation responses are predicted correctly for 11 of 15 patients (79%), with a positive predicted value of 0.85. Patients that are predicted incorrectly (False Positive, N=2; False Negative, N=2) had a higher PSI than correctly predicted patients (mean 0.98 vs. 0.77, p=0.07). The two patients (13%) that did not achieve robust mid-treatment responses had PSI>0.95. The PSI model with trained tumor growth and radiation response rates can predict mid-treatment responses with excellent accuracy for PSI <0.95. The evaluated data set had insufficient number of patients with higher PSI. An independent data set that includes additional patients that did not achieve robust mid-treatment responses is required to fully evaluate the predictive power of PSI for OPC radiation response.

Impact of Cancer-Associated Fibroblast on the Radiation-Response of Solid Xenograft Tumors.

Increasing evidence indicates that the heterogeneous tumor stroma supports therapy resistance at multiple levels. Fibroblasts, particularly cancer-associated fibroblasts (CAFs) are critical components of the tumor stroma. However, the impact of CAFs on the outcome of radiotherapy (RT) is poorly understood. Here, we investigated if and how fibroblasts/CAFs modulate the radiation response of malignant tumors by altering cancer cell radiosensitivity or radioresistance in vitro and in vivo. The influence of fibroblasts on cancer cell proliferation, cell death induction and long-term survival after RT was studied using different sets of fibroblasts and cancer cells in an indirect co-culture (2D) system to analyse potential paracrine interactions or a 3D model to study direct interactions. Paracrine signals from embryonic NIH-3T3 fibroblasts promoted MPR31.4 prostate and Py8119 breast cancer cell proliferation. Indirect co-culture with L929 skin fibroblasts induced higher levels of apoptosis in irradiated MPR31.4 cells, while they promoted proliferation of irradiated Py8119 cells. In addition, NIH-3T3 fibroblasts promoted long-term clonogenic survival of both tumor cell types upon irradiation in the 3D co-culture system when compared to non-irradiated controls. Also in vivo, co-implantation of cancer cells and fibroblasts resulted in different effects depending on the respective cell combinations used: co-implantation of MPR31.4 cells with NIH-3T3 fibroblasts or of Py8119 cells with L929 fibroblasts led to increased tumor growth and reduced radiation-induced tumor growth delay when compared to the respective tumors without co-implanted fibroblasts. Taken together, the impact of fibroblasts on cancer cell behavior and radiation sensitivity largely depended on the respective cell types used as they either exerted a pro-tumorigenic and radioresistance-promoting effect, an anti-tumorigenic effect, or no effect. We conclude that the plasticity of fibroblasts allows for such a broad spectrum of activities by the same fibroblast and that this plasticity is at least in part mediated by cancer cell-induced fibroblast activation toward CAFs.

Auranofin Protects Intestine against Radiation Injury by Modulating p53/p21 Pathway and Radiosensitizes Human Colon Tumor.

The radiosensitivity of the normal intestinal epithelium is the major limiting factor for definitive radiotherapy against abdominal malignancies. Radiosensitizers, which can be used without augmenting radiation toxicity to normal tissue, are still an unmet need. Inhibition of proteosomal degradation is being developed as a major therapeutic strategy for anticancer therapy as cancer cells are more susceptible to proteasomal inhibition-induced cytotoxicity compared with normal cells. Auranofin, a gold-containing antirheumatoid drug, blocks proteosomal degradation by inhibiting deubiquitinase inhibitors. In this study, we have examined whether auranofin selectively radiosensitizes colon tumors without promoting radiation toxicity in normal intestine. The effect of auranofin (10 mg/kg i.p.) on the radiation response of subcutaneous CT26 colon tumors and the normal gastrointestinal epithelium was determined using a mouse model of abdominal radiation. The effect of auranofin was also examined in a paired human colonic organoid system using malignant and nonmalignant tissues from the same patient. Both in the mouse model of intestinal injury and in the human nonmalignant colon organoid culture, auranofin pretreatment prevented radiation toxicity and improved survival with the activation of p53/p21-mediated reversible cell-cycle arrest. However, in a mouse model of abdominal tumor and in human malignant colonic organoids, auranofin inhibited malignant tissue growth with inhibition of proteosomal degradation, induction of endoplasmic reticulum stress/unfolded protein response, and apoptosis. Our data suggest that auranofin is a potential candidate to be considered as a combination therapy with radiation to improve therapeutic efficacy against abdominal malignancies.

Abstract 2922: The radioprotector GC4419 enhances the response of squamous cell carcinoma of the head and neck tumors to ionizing radiation and enhances radioimmune therapy

Abstract Locoregional recurrence (LRR) is the major cause of morbidity and mortality in patients with squamous cell carcinoma of the head and neck (HNSCC). For those patients that experience surgically unresectable LRR, median overall survival (OS) is less than one year. Approaches utilizing stereotactic ablative radiotherapy (SAbR) and immune oncology agents such as Nivolumab (α-PD-1 agonist) in the salvage setting have become alternatives to conventional chemoradiation therapy with one randomized, Phase II clinical trial combining these two modalities currently recruiting (clinicaltrials.gov identifier: NCT02684253). Despite improvements in OS, rates of acute oral mucositis (OM) and late toxicities (skin fibrosis) remain a concern with SAbR, particularly in the context of re-irradiation. Here we present pre-clinical evidence that GC4419 (Galera Therapeutics), a highly selective superoxide dismutase mimetic that recently received FDA Breakthrough therapy status following a Phase II, randomized, placebo controlled trial for reducing the duration and incidence of severe OM in patients undergoing chemoradiation therapy for locally advanced HNSCC (NCT02508389) also enhances the anti-tumor radiation response of HNSCC tumors to irradiation. GC4419 slows the growth of a panel of HNSCC cell lines in vitro, and utilizing clonogenic survival assays, demonstrates single agent anti-cancer activity at physiologically achievable concentrations, while also enhancing the response of HNSCC lines to irradiation. In tumor growth delay (TGD) experiments utilizing the syngeneic HNSCC tumor model, AT-84, when GC4419 is delivered starting 30-60 minutes prior to irradiation with biologically equivalent fractionation schedules of 17 Gy x 1 fxn, 10.24 Gy x 2 fxn, or 5 Gy x 5 fxn, a significant enhancement of the radiation response is achieved. Furthermore, when GC4419 is combined with radiation and α-PD-1 inhibitor, a further enhancement of the radiation response is observed, indicating that GC4419 compliments radiotherapy combined with immune oncology therapies. This potentiation of the anti-tumor response with GC4419 is more pronounced at doses that exceed the threshold to be considered for intensity modulated radiation therapy (IMRT). Results from clinical trials and the accompanying pre-clinical data strongly suggest that GC4419 should be combined with radio-immune therapy to not only enhance local tumor control, but that the potential for normal tissue protection with SAbR also creates the opportunity for dose escalation and may further improvement in treatment outcome. Citation Format: Brock J. Sishc, Elizabeth M. Polsdofer, Yuanyuan Zhang, Debabrata Saha, Michael D. Story. The radioprotector GC4419 enhances the response of squamous cell carcinoma of the head and neck tumors to ionizing radiation and enhances radioimmune therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2922.

Exosomal microRNA-199b-5p as a potential circulating biomarker to predict response of preoperative chemoradiotherapy for locally advanced rectal cancer.

e15161 Background: Preoperative chemoradiotherapy (CRT) followed by total mesorectal excision has become the standard treatment for locally advanced rectal cancer (LARC). However, the effect of CRT varies from complete response to complete resistance, and predicting response to CRT have not been well characterized yet. Previous studies have shown the potential of micro-RNA (miRNA) based approaches to enhance tumor radiation response. Accordingly, the present study attempted to identify biomarkers to predict response for preoperative CRT using comprehensive miRNA analysis in patients with LARC. Methods: This study included 65 rectal cancer tissues and 90 serum samples from patients who diagnosed with LACR and received preoperative CRT at Kyungpook National University Chilgok Hospital. Tissue specimens and serum samples were collected before CRT to evaluate the biologic differences between the good CRT response group and the poor CRT response group. For discovery of specific miRNAs, 800 miRNAs were analyzed using NanoString in 30 rectal cancer tissues. Thereafter, a total of 65 tissues, and 90 serum samples were investigated using real-time PCR for validation. Results: The median age was 59 years (range, 30-82), and the ratio of male to female was 3.05 to 1. The pathologic stages after preoperative CRT were as follows: pathologic complete response (n=13, 14.4%), pathologic stage I (n=13, 14.4%), pathologic stage II (n=27, 30.0%), pathologic stage III (n=28, 31.1%), and pathologic stage IV (n=9, 10.0%). In the discovery set, 16 target miRNAs were detected. In the validation set with tissue specimens, expression of 3 miRNAs (miR-199a/b-3p ( p=0.032), miR-199a-5p ( p=0.023), miR-199b-5p ( p=0.005)) was significantly upregulated which was associated with better response of CRT. Moreover, among the 3 candidate miRNAs, miR-199b-5p level was significantly upregulated in serum, and it was also found to be related with better response of CRT in LARC (pathologic stage 0/I versus II/III/IV, p=0.027). Conclusions: In the present study, high level of exosomal miR-199b-5p was associated with better response, suggesting it to be a promising non-invasive biomarker to predict response of CRT in patients with LARC. Accordingly, specific miRNAs can be predictive biomarker or therapeutic target to overcome radiotherapy resistance in LARC with a functional study.

The immune mediated role of extracellular HMGB1 in a heterotopic model of bladder cancer radioresistance

Radical cystectomy (RC) together with bilateral pelvic lymph node dissection remains the standard treatment for muscle invasive bladder cancer (MIBC). However, radiation-based treatments such as tri-modal therapy (TMT) involving maximally performed transurethral resection of bladder tumor (TURBT), radiotherapy (XRT), and a chemosensitizer represent an attractive, less invasive alternative. Nevertheless, 25–30% of MIBC patients will experience local recurrence after TMT and half will develop metastasis. Radioresistance of tumor cells could potentially be one of the causes for local recurrence post treatment. High mobility group box-1 (HMGB1) was shown to play a role in bladder cancer radioresistance through its intracellular functions in promoting DNA damage repair and autophagy. Recently, HMGB1 was found to be passively released from irradiated tumor cells. However, less is known about the involvement of extracellular HMGB1 in impairing radiation response and its exact role in modulating the tumor immune microenvironment after XRT. We identified a novel mechanism of bladder cancer radioresistance mediated by the immunological functions of HMGB1. The combination of radiation plus extracellular HMGB1 inhibition markedly improved the radiation response of tumors and resulted in marked changes in the immune landscape. Moreover, combining radiation and HMGB1 inhibition significantly impaired tumor infiltrating MDSCs and TAMs -but not Tregs- and shifted the overall tumor immune balance towards anti-tumoral response. We conclude that extracellular HMGB1 is involved in bladder cancer radioresistance through promoting pro-tumor immune mechanisms.

Proliferation saturation index in an adaptive Bayesian approach to predict patient-specific radiotherapy responses

Purpose: Radiotherapy prescription dose and dose fractionation protocols vary little between individual patients having the same tumor grade and stage. To personalize radiotherapy a predictive model is needed to simulate radiation response. Previous modeling attempts with multiple variables and parameters have been shown to yield excellent data fits at the cost of non-identifiability and clinically unrealistic results.Materials and methods: We develop a mathematical model based on a proliferation saturation index (PSI) that is a measurement of pre-treatment tumor volume-to-carrying capacity ratio that modulates intrinsic tumor growth and radiation response rates. In an adaptive Bayesian approach, we utilize an increasing number of data points for individual patients to predict patient-specific responses to subsequent radiation doses.Results: Model analysis shows that using PSI as the only patient-specific parameter, model simulations can fit longitudinal clinical data with high accuracy (R2=0.84). By analyzing tumor response to radiation using daily CT scans early in the treatment, response to the remaining treatment fractions can be predicted after two weeks with high accuracy (c-index = 0.89).Conclusion: The PSI model may be suited to forecast treatment response for individual patients and offers actionable decision points for mid-treatment protocol adaptation. The presented work provides an actionable image-derived biomarker prior to and during therapy to personalize and adapt radiotherapy.

Progression-Related Loss of Stromal Caveolin 1 Levels Mediates Radiation Resistance in Prostate Carcinoma via the Apoptosis Inhibitor TRIAP1.

Tumour resistance to chemo- and radiotherapy, as well as molecularly targeted therapies, limits the effectiveness of current cancer treatments. We previously reported that the radiation response of human prostate tumours is critically regulated by CAV1 expression in stromal fibroblasts and that loss of stromal CAV1 expression in advanced tumour stages may contribute to tumour radiotherapy resistance. Here we investigated whether fibroblast secreted anti-apoptotic proteins could induce radiation resistance of prostate cancer cells in a CAV1-dependent manner and identified TRIAP1 (TP53 Regulated Inhibitor of Apoptosis 1) as a resistance-promoting CAV1-dependent factor. TRIAP1 expression and secretion was significantly higher in CAV1-deficient fibroblasts and secreted TRIAP1 was able to induce radiation resistance of PC3 and LNCaP prostate cancer cells in vitro, as well as of PC3 prostate xenografts derived from co-implantation of PC3 cells with TRIAP1-expressing fibroblasts in vivo. Immunohistochemical analyses of irradiated PC3 xenograft tumours, as well as of human prostate tissue specimen, confirmed that the characteristic alterations in stromal-epithelial CAV1 expression were accompanied by increased TRIAP1 levels after radiation in xenograft tumours and within advanced prostate cancer tissues, potentially mediating resistance to radiation treatment. In conclusion, we have determined the role of CAV1 alterations potentially induced by the CAV1-deficient, and more reactive, stroma in radio sensitivity of prostate carcinoma at a molecular level. We suggest that blocking TRIAP1 activity and thus avoiding drug resistance may offer a promising drug development strategy for inhibiting resistance-promoting CAV1-dependent signals.

Iron-oxide nanoparticles target intracellular HSP90 to induce tumor radio-sensitization

BackgroundNanoparticle-based therapies have emerged as a promising approach to overcome limitations of conventional chemotherapy. Present study investigates the potential of oleic acid-functionalized iron-oxide nanoparticles (MN-OA) to enhance the radiation response of fibrosarcoma tumor and elucidates its underlying mechanism. MethodsVarious cellular and molecular assays (e.g. MTT, clonogenic, cell cycle analysis, cell death, DNA damage/repair) and tumor growth kinetics were employed to investigate the mechanism of MN-OA induced radio-sensitization. ResultsMouse (WEHI-164) and human (HT-1080) fibrosarcoma cells treated with MN-OA and gamma-radiation (2 Gy) showed a significant decrease in the cell proliferation. Combination treatment showed significant decrease in clonogenic survival of WEHI-164 cells and was found to induce cell cycle arrest, apoptosis and mitotic catastrophe. The mechanism of radio-sensitization was found to involve binding of MN-OA with HSP90, resulting in down-regulation of its client proteins, involved in cell cycle progression (Cyclin B1 and CDC2) and DNA-double strand break repair (e.g. RAD51 and BRCA1). Consistently, longer persistence of DNA damage in cells treated with MN-OA and radiation was observed in the form of γ-H2AX foci. The efficacy and mechanism of MN-OA-induced radio-sensitization was also validated in an immuno-competent murine fibrosarcoma model. ConclusionThis study reveals the key role of HSP90 in the mechanism of tumor radio-sensitization by MN-OA. General significancePresent work provides a deeper understanding about the mechanism of MN-OA-induced tumor radiosensitization, highlighting the role of HSP90 protein. In addition to diagnostic and magnetic hyperthermia abilities, present remarkable radiosensitizing activity of MN-OA would further excite the clinicians to test its anti-cancer potential.