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Avasopasem manganese (AVA or GC4419), a selective superoxide dismutase mimetic, is in a phase 3 clinical trial (NCT03689712) as a mitigator of radiation-induced mucositis in head and neck cancer based on its superoxide scavenging activity. We tested whether AVA synergized with radiation via the generation of hydrogen peroxide, the product of superoxide dismutation, to target tumor cells in preclinical xenograft models of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, and pancreatic ductal adenocarcinoma. Treatment synergy with AVA and high dose per fraction radiation occurred when mice were given AVA once before tumor irradiation and further increased when AVA was given before and for 4 days after radiation, supporting a role for oxidative metabolism. This synergy was abrogated by conditional overexpression of catalase in the tumors. In addition, in vitro NSCLC and mammary adenocarcinoma models showed that AVA increased intracellular hydrogen peroxide concentrations and buthionine sulfoximine- and auranofin-induced inhibition of glutathione- and thioredoxin-dependent hydrogen peroxide metabolism selectively enhanced AVA-induced killing of cancer cells compared to normal cells. Gene expression in irradiated tumors treated with AVA suggested that increased inflammatory, TNFα, and apoptosis signaling also contributed to treatment synergy. These results support the hypothesis that AVA, although reducing radiotherapy damage to normal tissues, acts synergistically only with high dose per fraction radiation regimens analogous to stereotactic ablative body radiotherapy against tumors by a hydrogen peroxide-dependent mechanism. This tumoricidal synergy is now being tested in a phase I-II clinical trial in humans (NCT03340974)., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

EGFR signaling has been implicated in hypoxia-associated resistance to radiation or chemotherapy. Non-small cell lung carcinomas (NSCLC) with activating L858R or ΔE746-E750 EGFR mutations exhibit elevated EGFR activity and downstream signaling. Here, relative to wild-type (WT) EGFR, mutant (MT) EGFR expression significantly increases radiosensitivity in hypoxic cells. Gene expression profiling in human bronchial epithelial cells (HBEC) revealed that MT-EGFR expression elevated transcripts related to cell cycle and replication in aerobic and hypoxic conditions and downregulated RAD50, a critical component of nonhomologous end joining and homologous recombination DNA repair pathways. NSCLCs and HBEC with MT-EGFR revealed elevated basal and hypoxia-induced γ-H2AX-associated DNA lesions that were coincident with replication protein A in the S-phase nuclei. DNA fiber analysis showed that, relative to WT-EGFR, MT-EGFR NSCLCs harbored significantly higher levels of stalled replication forks and decreased fork velocities in aerobic and hypoxic conditions. EGFR blockade by cetuximab significantly increased radiosensitivity in hypoxic cells, recapitulating MT-EGFR expression and closely resembling synthetic lethality of PARP inhibition. Implications: This study demonstrates that within an altered DNA damage response of hypoxic NSCLC cells, mutant EGFR expression, or EGFR blockade by cetuximab exerts a synthetic lethality effect and significantly compromises radiation resistance in hypoxic tumor cells. Mol Cancer Res; 15(11); 1503-16. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The use of tumor-treating fields (TTFields) has revolutionized the treatment of recurrent and newly diagnosed glioblastoma (GBM). TTFields are low-intensity, intermediate frequency, alternating electric fields that are applied to tumor regions and cells using non-invasive arrays. The predominant mechanism by which TTFields are thought to kill tumor cells is the disruption of mitosis. Using five non-small cell lung cancer (NSCLC) cell lines we found that there is a variable response in cell proliferation and cell killing between these NSCLC cell lines that was independent of p53 status. TTFields treatment increased the G2/M population, with a concomitant reduction in S-phase cells followed by the appearance of a sub-G1 population indicative of apoptosis. Temporal changes in gene expression during TTFields exposure was evaluated to identify molecular signaling changes underlying the differential TTFields response. The most differentially expressed genes were associated with the cell cycle and cell proliferation pathways. However, the expression of genes found within the BRCA1 DNA-damage response were significantly downregulated (P<0.05) during TTFields treatment. DNA double-strand break (DSB) repair foci increased when cells were exposed to TTFields as did the appearance of chromatid-type aberrations, suggesting an interphase mechanism responsible for cell death involving DNA repair. Exposing cells to TTFields immediately following ionizing radiation resulted in increased chromatid aberrations and a reduced capacity to repair DNA DSBs, which were likely responsible for at least a portion of the enhanced cell killing seen with the combination. These findings suggest that TTFields induce a state of 'BRCAness' leading to a conditional susceptibility resulting in enhanced sensitivity to ionizing radiation and provides a strong rationale for the use of TTFields as a combined modality therapy with radiation or other DNA-damaging agents.

Stereotactic body radiation therapy (SBRT) has found an important role in the treatment of patients with non-small cell lung cancer, demonstrating improvements in dose distribution and even tumor cure rates, particularly for early-stage disease. Despite its emerging clinical efficacy, SBRT has primarily evolved due to advances in medical imaging and more accurate dose delivery, leaving a void in knowledge of the fundamental biological mechanisms underlying its activity. Thus, there is a critical need for the development of orthotropic animal models to further probe the biology associated with high-dose-per-fraction treatment typical of SBRT. We report here on an improved surgically based methodology for generating solitary intrapulmonary nodule tumors, which can be treated with simulated SBRT using the X-RAD 225Cx small animal irradiator and Small Animal RadioTherapy (SmART) Plan treatment system. Over 90% of rats developed solitary tumors in the right lung. Furthermore, the tumor response to radiation was monitored noninvasively via bioluminescence imaging (BLI), and complete ablation of tumor growth was achieved with 36 Gy (3 fractions of 12 Gy each). We report a reproducible, orthotopic, clinically relevant lung tumor model, which better mimics patient treatment regimens. This system can be utilized to further explore the underlying biological mechanisms relevant to SBRT and high-dose-per-fraction radiation exposure and to provide a useful model to explore the efficacy of radiation modifiers in the treatment of non-small cell lung cancer.

Therapeutic decisions in non-small cell lung cancer (NSCLC) have been mainly based on disease stage, performance status, and co-morbidities, and rarely on histological or molecular classification. Rather than applying broad treatments to unselected patients that may result in survival increase of only weeks to months, research efforts should be, and are being, focused on identifying predictive markers for molecularly targeted therapy and determining genomic signatures that predict survival and response to specific therapies. The availability of such targeted biologics requires their use to be matched to tumors of corresponding molecular vulnerability for maximum efficacy. Molecular markers such as epidermal growth factor receptor (EGFR), K-ras, vascular endothelial growth factor (VEGF), mammalian target of rapamycin (mTOR), and anaplastic lymphoma kinase (ALK) represent potential parameters guide treatment decisions. Ultimately, identifying patients who will respond to specific therapies will allow optimal efficacy with minimal toxicity, which will result in more judicious and effective application of expensive targeted therapy as the new paradigm of personalized medicine develops., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The recently developed ability to interrogate genome-wide data arrays has provided invaluable insights into the molecular pathogenesis of lung cancer. These data have also provided information for developing targeted therapy in lung cancer patients based on the identification of cancer-specific vulnerabilities and set the stage for molecular biomarkers that provide information on clinical outcome and response to treatment. In addition, there are now large panels of lung cancer cell lines, both non-small-cell lung cancer and small-cell lung cancer, that have distinct chemotherapy and radiation response phenotypes. We anticipate that the integration of molecular data with therapy response data will allow for the generation of biomarker signatures that predict response to therapy. These signatures will need to be validated in clinical studies, at first retrospective analyses and then prospective clinical trials, to show that the use of these biomarkers can aid in predicting patient outcomes (eg, in the case of radiation therapy for local control and survival). This review highlights recent advances in molecular profiling of tumor responses to radiotherapy and identifies challenges and opportunities in developing molecular biomarker signatures for predicting radiation response for individual patients with lung cancer., (Copyright 2010 Elsevier Inc. All rights reserved.)

The purpose of this study was to develop a rat orthotopic lung tumor model with a solitary intrapulmonary nodule to study the effects of high-dose radiation. A549-Luc non-small cell lung cancer (NSCLC) cells were implanted into nude rats in the intercostal space between ribs 5 and 6 of the right lung. Bioluminescence and microcomputed tomography (CT) imaging were performed after implantation to confirm the presence of a solitary tumor and to monitor tumor growth. A device using image guidance for localization was developed to facilitate high-precision irradiation in small animals. A pilot irradiation study was performed, and response was assessed by bioluminescence imaging and immunohistochemistry. Radiation response was confirmed through serial bioluminescence imaging, and the strength of the bioluminescence signal was observed to be inversely proportional to dose. Response was also observed by the monoclonal antibody bavituximab, which binds to exposed lipid phosphatidylserine (PS) on tumor vessels. The ability to (1) reproducibly generate solitary tumor nodules in the rat lung, (2) identify and monitor tumor growth by bioluminescence imaging and CT imaging, (3) accurately target these tumors using high doses of radiation, and (4) demonstrate and quantify radiation response using bioluminescence imaging provides significant opportunity to probe the biological mechanisms of high-dose irradiation in preclinical settings.

In solid tumors, including non-small cell lung carcinomas (NSCLC) the existence of radioresistant subpopulations, such as quiescent or hypoxic tumor cells, is well established, thus posing a critical therapeutic problem. Although small-molecule inhibitors targeting cyclin-dependent kinases (CDK) were demonstrated to enhance cellular radiosensitivity preferentially in proliferating tumor cells, cell cycle-independent activities of these substances were recently suggested. In this study, the potential of a newer generation small-molecule CDK inhibitor, SNS-032, to sensitize radioresistant tumor cells to ionizing radiation was tested in vitro using two NSCLC cell lines (NCI-H460 and A549). Exposure of quiescent and hypoxic lung tumor cells to SNS-032 at a clinically achievable concentration (500 nM) prior to irradiation resulted in a significant increase in cellular radiosensitivity indicating cell cycle-unrelated mechanisms. The effect of SNS-032 on non-cycling cells was not attributed to an enhanced toxicity of the drug. A SNS-032 mediated delay in the resolution of radiation-induced gammaH2AX foci a surrogate for DNA double-strand breaks was determined in non-cycling cells, suggesting a modulation of DNA double-strand break repair. These results indicate a modulation of DNA double-strand break repair to be partially attributed to the radiosensitization effects of SNS-032 observed in hypoxic and quiescent lung tumor cells. Considering the importance of therapy resistance for the radiocurability of solid tumors, our findings may provide the basis for an improvement of the well-established treatment regimens in clinical oncology.

The epidermal growth factor receptor (EGFR) is an important determinant of radioresponse, whose elevated expression and activity frequently correlates with radioresistance in several cancers, including non-small cell lung carcinoma (NSCLC). We reported recently that NSCLC cell lines harboring somatic, activating mutations in the tyrosine kinase domain (TKD) of the EGFR exhibit significant delays in the repair of DNA double-strand breaks (DSB) and poor clonogenic survival in response to radiation. Here, we explore the mechanisms underlying mutant EGFR-associated radiosensitivity. In three representative NSCLC cell lines, we show that, unlike wild-type (WT) EGFR, receptors with common oncogenic TKD mutations, L858R or DeltaE746-E750, are defective in radiation-induced translocation to the nucleus and fail to bind the catalytic and regulatory subunits of the DNA-dependent protein kinase (DNA-PK), a key enzyme in the nonhomologous end-joining repair pathway. Moreover, despite the presence of WT EGFR, stable exogenous expression of either the L858R or the DeltaE746-E750 mutant forms of EGFR in human bronchial epithelial cells significantly delays repair of ionizing radiation (IR)-induced DSBs, blocks the resolution of frank or microhomologous DNA ends, and abrogates IR-induced nuclear EGFR translocation or binding to DNA-PK catalytic subunit. Our study has identified a subset of naturally occurring EGFR mutations that lack a critical radioprotective function of EGFR, providing valuable insights on how the EGFR mediates cell survival in response to radiation in NSCLC cell lines.

Non-small cell lung cancers (NSCLCs) bearing mutations in the tyrosine kinase domain (TKD) of the epidermal growth factor receptor (EGFR) often exhibit dramatic sensitivity to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Ionizing radiation (IR) is frequently used in the treatment of NSCLC, but little is known how lung tumor-acquired EGFR mutations affect responses to IR. Because this is of great clinical importance, we investigated and found that clonogenic survival of mutant EGFR NSCLCs in response to IR was reduced 500- to 1,000-fold compared with wild-type (WT) EGFR NSCLCs. Exogenous expression of either the L858R point mutant or the DeltaE746-E750 deletion mutant form of EGFR in immortalized human bronchial epithelial cells, p53 WT NSCLC (A549), or p53-null NSCLC (NCI-H1299) resulted in dramatically increased sensitivity to IR. We show that the majority of mutant EGFR NSCLCs, including those that contain the secondary gefitinib resistance T790M mutation, exhibit characteristics consistent with a radiosensitive phenotype, which include delayed DNA repair kinetics, defective IR-induced arrest in DNA synthesis or mitosis, and pronounced increases in apoptosis or micronuclei. Thus, understanding how activating mutations in the TKD domain of EGFR contribute to radiosensitivity should provide new insight into effective treatment of NSCLC with radiotherapy and perhaps avoid emergence of single agent drug resistance.