G-CSF rescues tumor growth and neo-angiogenesis during liver metastasis under host angiopoietin-2 deficiency.

Suppression of neo-angiogenesis is a clinically used anti-tumor strategy with new targets such as angiopoietin-2 (Ang2) being proposed. However, the functions of Ang2 in vascular remodeling, inflammation and tumor growth are not consistent. We examined effect of depletion of host Ang2 on liver colony formation using Ang2 deficient (Ang2(-/-)) mice. Surprisingly, the metastatic colonies formed in Ang2(-/-) mice were larger than those in the wild type. These colonies had greater vascular density with more pericyte coverage than the vessels in liver colonies in the wild type. Liver VEGF concentration in both genotypes was equivalent, and thus, the differences appeared VEGF independent. However, after colony formation, the serum concentration of granulocyte-colony stimulating factor (G-CSF) and CXCL1 in Ang2(-/-) mice was 12 and 6 times greater than after colony formation in wild type. Increase of these two cytokines was associated with two times greater numbers of neutrophils recruited to the liver. Two times more Tie2+/CD11b+/CD31- cells were present in the tumors in Ang2(-/-) than in the wild type livers. These results suggest that the depletion of host Ang2 induced compensatory VEGF-independent angiogenic mechanisms and thus enhanced liver metastatic colony growth and colony vascularity. They further indicate organotypic differences in response to tumor metastasis. In contrast, Ang2 deficiency inhibited tumor growth during metastatic colony formation in the lung, consistent with the reports of decreased pulmonary seeding of tumor cells after pharmacological inhibition of Ang2. Further studies are thus required to assess the effects of pharmacological Ang2 blockade for cancer patients particularly in the liver.

Bnip3L is induced by p53 under hypoxia, and its knockdown promotes tumor growth.

p53-dependent apoptosis is a major determinant of its tumor suppressor activity and can be triggered by hypoxia. No p53 target is known to be induced by p53 or to mediate p53-dependent apoptosis during hypoxia. We report that p53 can directly upregulate expression of Bnip3L, a cell death inducer. During hypoxia, Bnip3L is highly induced in wild-type p53-expressing cells, in part due to increased recruitment of p53 and CBP to Bnip3L. Apoptosis is reduced in hypoxia-exposed cells with functional p53 following Bnip3L knockdown. In vivo, Bnip3L knockdown promotes tumorigenicity of wild-type versus mutant p53-expressing tumors. Thus, Bnip3L, capable of attenuating tumorigenicity, mediates p53-dependent apoptosis under hypoxia, which provides a novel understanding of p53 in tumor suppression.

BID regulation by p53 contributes to chemosensitivity.

The role of the p53 protein (encoded by TP53) in tumour suppression relies partly on the ability of p53 to regulate the transcription of genes that are important in cell-cycle arrest and in apoptosis. But the apoptotic pathway mediated by p53 is not fully understood. Here we show that BID, a member of the pro-apoptotic Bcl-2 family of proteins, is regulated by p53. BID mRNA is increased in a p53-dependent manner in vitro and in vivo, with strong expression in the splenic red pulp and colonic epithelium of gamma-irradiated mice. Both the human and the mouse BID genomic loci contain p53-binding DNA response elements that bind p53 and mediate p53-dependent transactivation of a reporter gene. In addition, BID-null mouse embryonic fibroblasts are more resistant than are wild-type fibroblasts to the DNA damaging agent adriamycin and the nucleotide analogue 5-fluorouracil, both of which stabilize endogenous p53. Our results indicate that BID is a p53-responsive 'chemosensitivity gene' that may enhance the cell death response to chemotherapy.

Overview and Lessons From the Preclinical Chemoradiotherapy Testing Consortium.

PURPOSE: In the current molecular-targeted cancer treatment era, many new agents are being developed so that optimizing therapy with a combination of radiation and drugs is complex. The use of emerging laboratory technologies to further biological understanding of drug-radiation mechanisms of action will enhance the efficiency of the progression from preclinical studies to clinical trials. In 2017, the National Cancer Institute (NCI) solicited proposals through PAR 16-111 to conduct preclinical research combining targeted anticancer agents in the Cancer Therapy Evaluation Program's portfolio with chemoradiation. METHODS AND MATERIALS: The Preclinical Chemo-Radiotherapy Testing Consortium (PCRTC) was formed with 4 U01 programs supported to generate validated high-quality preclinical data on the effects of molecular therapeutics when added to standard-of-care therapies with a concentration on cancers of the pancreas, lung, head and neck, gastrointestinal tract, and brain. RESULTS: The PCRTC provides a rational basis for prioritizing NCI-supported investigational new drugs or agents most likely to have clinical activity with chemoradiotherapy and accelerate the pace at which combined modality treatments with greater efficacy are identified and incorporated into standard treatment practices. CONCLUSIONS: Herein, we introduce and summarize the course of the PCRTC to date and report 3 preliminary observations from the consortium's work to date.

SMAC Mimetic/IAP Inhibitor Birinapant Enhances Radiosensitivity of Glioblastoma Multiforme.

Birinapant is a novel SMAC peptidomimetic molecule in clinical development. It suppresses the inhibitor of apoptosis proteins (IAPs) and promotes cytochrome-C/Apaf-1/caspase-9 activation to induce effective apoptosis. Because IAP inhibition has been shown to enhance the sensitivity of cancer cells to radiation, we investigated the role of birinapant in radiosensitization of glioblastoma cells in vitro and in vivo. Two glioblastoma cell lines, U-251 and U-87, were used to analyze radiosensitization in vitro with 7-AAD cell death/apoptosis and clonogenic assays. Subcutaneous flank (U-251 and U-87) and intracranial orthotopic (U-251) xenografts in nude mice were used to evaluate radiosensitization in vivo. TNF-α levels in media and serum were measured using electrochemiluminescence. Radiosensitization in vitro was more prominent for U-251 cells than for U-87 cells. In vivo, in both tumor models, significant tumor growth delay was observed with combination treatment compared to radiation alone. There was a survival benefit with combination treatment in the orthotopic U-251 model. TNF-α levels in media correlated directly with radiation dose in vitro. These findings show that birinapant can enhance the radiosensitivity of glioblastoma cell lines in cell-based assays and tumor models via radiation-induced TNF-α. Further study into the use of birinapant with radiation therapy is warranted.

Phosphatase and tensin homologue deficiency in glioblastoma confers resistance to radiation and temozolomide that is reversed by the protease inhibitor nelfinavir.

Glioblastomas are malignant brain tumors that are very difficult to cure, even with aggressive therapy consisting of surgery, chemotherapy, and radiation. Glioblastomas frequently have loss of the phosphatase and tensin homologue (PTEN), leading to the activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway. We examined whether PTEN deficiency leads to radioresistance and whether this can be reversed by nelfinavir, a protease inhibitor that decreases Akt signaling. Nelfinavir decreased Akt phosphorylation and enhanced radiosensitization in U251MG and U87MG glioblastoma cells, both of which are PTEN deficient. In the derivative line U251MG-PTEN, induction of wild-type PTEN with doxycycline decreased P-Akt expression and increased radiosensitivity to a similar extent as nelfinavir. Combining these two approaches had no greater effect on radiosensitivity than either alone. This epistasis-type analysis suggests that the nelfinavir acts along the Akt pathway to radiosensitize cells. However, nelfinavir neither decreased Akt phosphorylation in immortalized human astrocytes nor radiosensitized them. Radiosensitization was also assessed in vivo using a tumor regrowth delay assay in nude mice implanted with U87MG xenografts. The mean time to reach 1,000 mm(3) in the radiation + nelfinavir group was 71 days, as compared with 41, 34, or 45 days for control, nelfinavir alone, or radiation alone groups, respectively. A significant synergistic effect on tumor regrowth was detected between radiation and nelfinavir. (P = 0.01). Nelfinavir also increased the sensitivity of U251MG cells to temozolomide. These results support the clinical investigation of nelfinavir in combination with radiation and temozolomide in future clinical trials for patients with glioblastomas.

DR5 knockout mice are compromised in radiation-induced apoptosis.

DR5 (also called TRAIL receptor 2 and KILLER) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (also called TRAIL and Apo2 ligand). DR5 is a transcriptional target of p53, and its overexpression induces cell death in vitro. However, the in vivo biology of DR5 has remained largely unexplored. To better understand the role of DR5 in development and in adult tissues, we have created a knockout mouse lacking DR5. This mouse is viable and develops normally with the exception of having an enlarged thymus. We show that DR5 is not expressed in developing embryos but is present in the decidua and chorion early in development. DR5-null mouse embryo fibroblasts expressing E1A are resistant to treatment with TRAIL, suggesting that DR5 may be the primary proapoptotic receptor for TRAIL in the mouse. When exposed to ionizing radiation, DR5-null tissues exhibit reduced amounts of apoptosis compared to wild-type thymus, spleen, Peyer's patches, and the white matter of the brain. In the ileum, colon, and stomach, DR5 deficiency was associated with a subtle phenotype of radiation-induced cell death. These results indicate that DR5 has a limited role during embryogenesis and early stages of development but plays an organ-specific role in the response to DNA-damaging stimuli.

Nelfinavir down-regulates hypoxia-inducible factor 1alpha and VEGF expression and increases tumor oxygenation: implications for radiotherapy.

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway can increase vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha (HIF-1alpha) expression. We examined the effect of nelfinavir, an HIV protease inhibitor that inhibits Akt signaling, on VEGF and HIF-1alpha expression and on angiogenesis, tumor oxygenation, and radiosensitization. Nelfinavir decreases VEGF expression under normoxia via the transcription factor Sp1, which regulates the proximal core VEGF promoter. Nelfinavir decreased Sp1 phosphorylation and decreased Sp1 binding to a probe corresponding to the proximal VEGF promoter in a gel shift assay. Nelfinavir also decreased the hypoxic induction of HIF-1alpha, which also regulates the VEGF promoter, most likely by decreasing its translation. The effect of nelfinavir on VEGF expression had the functional consequence of decreasing angiogenesis in an in vivo Matrigel plug assay. To determine the effect this might have on tumor radiosensitization, we did tumor regrowth assays with xenografts in nude mice. The combination of nelfinavir and radiation increased time to regrowth compared with radiation alone whereas nelfinavir alone had little effect on tumor regrowth. This radiosensitizing effect was greater than suggested by in vitro clonogenic survival assays. One possible explanation for the discordance is that nelfinavir has an effect on tumor oxygenation. Therefore, we examined this with the hypoxia marker EF5 and found that nelfinavir leads to increased oxygenation within tumor xenografts. Our results suggest that nelfinavir decreases HIF-1alpha/VEGF expression and tumor hypoxia, which could play a role in its in vivo radiosensitizing effect. These data support the use of nelfinavir in combination with radiation in future clinical trials.

Circulating Tumor DNA Assays in Clinical Cancer Research.

The importance of circulating free DNA (cfDNA) in cancer clinical research was recognized in 1994 when a mutated RAS gene fragment was detected in a patient's blood sample. Up to 1% of the total circulating DNA in patients with cancer is circulating tumor DNA (ctDNA) that originates from tumor cells. As ctDNA is rapidly cleared from the blood stream and can be obtained by minimally invasive methods, it can be used as a dynamic cancer biomarker for cancer early detection, diagnosis, and treatment monitoring. Despite the potential for clinical use, few ctDNA assays have been cleared or approved by the US Food and Drug Administration. As tools for clinical and translational research, current ctDNA assays face some challenges, and more research is needed to advance use of these assays. On September 29-30, 2016, the Division of Cancer Treatment and Diagnosis at the National Cancer Institute convened a workshop entitled "Circulating Tumor DNA Assays in Clinical Cancer Research" to garner input from industry experts, academia, and government research and regulatory agencies to understand and promote the translation of ctDNA assays to clinical research, with potential to advance to use in clinical practice. This Commentary presents the topics of the workshop covered in the presentations and points made in the discussions that followed: 1) background on ctDNA, 2) potential clinical utility of ctDNA assays, 3) assay technology, 4) assay clinical and analytical validation, and 5) industry perspectives. Additional relevant information that has come to light since the workshop has been included.

The Future of Radiobiology.

Innovation and progress in radiation oncology depend on discovery and insights realized through research in radiation biology. Radiobiology research has led to fundamental scientific insights, from the discovery of stem/progenitor cells to the definition of signal transduction pathways activated by ionizing radiation that are now recognized as integral to the DNA damage response (DDR). Radiobiological discoveries are guiding clinical trials that test radiation therapy combined with inhibitors of the DDR kinases DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), ataxia telangiectasia related (ATR), and immune or cell cycle checkpoint inhibitors. To maintain scientific and clinical relevance, the field of radiation biology must overcome challenges in research workforce, training, and funding. The National Cancer Institute convened a workshop to discuss the role of radiobiology research and radiation biologists in the future scientific enterprise. Here, we review the discussions of current radiation oncology research approaches and areas of scientific focus considered important for rapid progress in radiation sciences and the continued contribution of radiobiology to radiation oncology and the broader biomedical research community.

Akt1 activation can augment hypoxia-inducible factor-1alpha expression by increasing protein translation through a mammalian target of rapamycin-independent pathway.

The phosphoinositide 3-kinase (PI3K)/Akt pathway is commonly activated in cancer; therefore, we investigated its role in hypoxia-inducible factor-1alpha (HIF-1alpha) regulation. Inhibition of PI3K in U87MG glioblastoma cells, which have activated PI3K/Akt activity secondary to phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation, with LY294002 blunted the induction of HIF-1alpha protein and its targets vascular endothelial growth factor and glut1 mRNA in response to hypoxia. Introduction of wild-type PTEN into these cells also blunted HIF-1alpha induction in response to hypoxia and decreased HIF-1alpha accumulation in the presence of the proteasomal inhibitor MG132. Akt small interfering RNA (siRNA) also decreased HIF-1alpha induction under hypoxia and its accumulation in normoxia in the presence of dimethyloxallyl glycine, a prolyl hydroxylase inhibitor that prevents HIF-1alpha degradation. Metabolic labeling studies showed that Akt siRNA decreased HIF-1alpha translation in normoxia in the presence of dimethyloxallyl glycine and in hypoxia. Inhibition of mammalian target of rapamycin (mTOR) with rapamycin (10-100 nmol/L) had no significant effect on HIF-1alpha induction in a variety of cell lines, a finding that was confirmed using mTOR siRNA. Furthermore, neither mTOR siRNA nor rapamycin decreased HIF-1alpha translation as determined by metabolic labeling studies. Therefore, our results indicate that Akt can augment HIF-1alpha expression by increasing its translation under both normoxic and hypoxic conditions; however, the pathway we are investigating seems to be rapamycin insensitive and mTOR independent. These observations, which were made on cells grown in standard tissue culture medium (10% serum), were confirmed in PC3 prostate carcinoma cells. We did find that rapamycin could decrease HIF-1alpha expression when cells were cultured in low serum, but this seems to represent a different pathway.

Sp1 is involved in Akt-mediated induction of VEGF expression through an HIF-1-independent mechanism.

Increased expression of vascular endothelial growth factor (VEGF) contributes to the growth of many tumors by increasing angiogenesis. Although hypoxia is a potent inducer of VEGF, we previously showed that epidermal growth factor receptor amplification and loss of PTEN, both of which can increase phosphatidylinositol-3-kinase (PI3K) activity, increase VEGF expression. Using both adenoviral vectors and a cell line permanently expressing constitutively active myristoylated Akt (myrAkt), we show that activation of Akt, which is downstream of PI3K, increases VEGF expression in vitro and increases angiogenesis in a Matrigel plug assay. Transient transfection experiments using reporter constructs containing the VEGF promoter showed that up-regulation of VEGF by Akt is mediated through Sp1 binding sites located in the proximal promoter. Small interfering RNA directed against Sp1 prevented the induction of VEGF mRNA in response to myrAkt but not to hypoxia. Expression of myrAkt is associated with increased phosphorylation of Sp1 and its increased binding to a probe corresponding to the -88/-66 promoter region. In conclusion, our results indicate that Sp1 is required for transactivation of the VEGF by Akt. Others have proposed that the PI3K/Akt pathway can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1); however, our results suggest an alternative mechanism can also operate.

Selective inhibition of Ras, phosphoinositide 3 kinase, and Akt isoforms increases the radiosensitivity of human carcinoma cell lines.

Ras activation promotes the survival of tumor cells after DNA damage. To reverse this survival advantage, Ras signaling has been targeted for inhibition. Other contributors to Ras-mediated DNA damage survival have been identified using pharmacologic inhibition of signaling, but this approach is limited by the specificity of the inhibitors used and their toxicity. To better define components of Ras signaling that could be inhibited in a clinical setting, RNA interference was used to selectively block expression of specific isoforms of Ras, phosphoinositide 3 (PI3) kinase, and Akt. Inhibition of oncogenic Ras expression decreased both phospho-Akt and phospho-p42/44 mitogen-activated protein (MAP) kinase levels and reduced clonogenic survival. Because pharmacologic inhibition of PI3 kinases and Akt radiosensitized cell lines with active Ras signaling, whereas inhibition of the MAP/extracellular signal-regulated kinase (ERK) kinase/ERK pathway did not, we examined the contribution of PI3 kinases and Akts to radiation survival. Selective inhibition the PI3 kinase P110alpha + p85beta isoforms reduced Akt phosphorylation and radiation survival. Similarly, inhibition of Akt-1 reduced tumor cell radiation survival. Inhibition of Akt-2 or Akt-3 had less effect. Retroviral transduction and overexpression of mouse Akt-1 was shown to rescue cells from inhibition of endogenous human Akt-1 expression. This study shows that Ras signaling to the PI3 kinase-Akt pathway is an important contributor to survival, whether Ras activation results from mutation of ras or overexpression of epidermal growth factor receptor. This study further shows that selective inhibition of the PI3 kinase P110alpha + p85beta isoforms or Akt-1 could be a viable approach to sensitizing many tumor cells to cytotoxic therapies.

Pancreatic cancer cell radiation survival and prenyltransferase inhibition: the role of K-Ras.

Activating K-ras mutations are found in approximately 90% of pancreatic carcinomas and may contribute to the poor prognosis of these tumors. Because radiotherapy is frequently used in pancreatic cancer treatment, we assessed the contribution of oncogenic K-ras signaling to pancreatic cancer radiosensitivity. Seven human pancreatic carcinoma lines with activated K-ras and two cell lines with wild-type ras were used to examine clonogenic cell survival after Ras inhibition. Ras inhibition was accomplished by small interfering RNA (siRNA) knockdown of K-ras expression and by blocking Ras processing using a panel of prenyltransferase inhibitors of differing specificity for the two prenyltransferases that modify K-Ras. K-ras knockdown by siRNA or inhibition of prenyltransferase activity resulted in radiation sensitization in vitro and in vivo in tumors with oncogenic K-ras mutations. Inhibition of farnesyltransferase alone was sufficient to radiosensitize most K-ras mutant tumors, although K-Ras prenylation was not blocked. These results show that inhibition of activated K-Ras can promote radiation killing of pancreatic carcinoma in a superadditive manner. The finding that farnesyltransferase inhibition alone radiosensitizes tumors with K-ras mutations implies that a farnesyltransferase inhibitor-sensitive protein other than K-Ras may contribute to survival in the context of mutant K-ras. Farnesyltransferase inhibitors could therefore be of use as sensitizers for pancreatic carcinoma radiotherapy.

Radiosensitizing effects of the prenyltransferase inhibitor AZD3409 against RAS mutated cell lines.

Mutations at the H-, N- and K-ras loci are among the most frequent genetic alterations in human cancers. In this study, we have investigated the effect of AZD3409, a novel, peptidomimetic prenyltransferase inhibitor (PTI), on the radiosensitivity of cells with mutated ras alleles. AZD3409, developed by AstraZeneca, inhibits both farnesyl- and geranylgeranyl transferase in cell free systems. AZD3409 inhibits the growth of a variety of human cancer cell lines, including cells that express mutant alleles of either K- or H- ras and was well tolerated when administered orally to healthy volunteers in a phase I clinical trial. We have previously shown that PTI can radiosensitize human and rodent cancer cell lines that express activated RAS. Here we assessed the ability of AZD3409 to radiosensitize human cancer cell lines in vivo and in vitro and the activation state of RAS proteins in treated cells. Once daily oral administration of AZD3409 to nude mice bearing PSN-1 and MiaPaCa-2 human pancreatic cancer xenografts expressing mutant K-ras was well tolerated and resulted in a supra-additive reduction in clonogenic cell survival after irradiation. Similarly, AZD3409 reduced clonogenic survival in cells that express either mutant K- or H- ras in vitro. We next examined the effect of AZD3409 on the processing and activation of K- and H-RAS. AZD3409-mediated radiosensitization, both in vivo and in vitro, correlates with a decrease in H-RAS processing without detectable effect on K-RAS processing. RAS activation assays show that the decreased H-RAS processing is accompanied by decreased H-RAS activation in cell lines with mutations in either K- or H-ras. However, no decrease in K-RAS activation was detected. Thus, radiosensitization of human cancer cells that express mutated K-RAS occurred under conditions where AZD3409 inihibits the activation of farneyslated H-RAS, but did not inhibit K-RAS activation.

Tissue-specific induction of p53 targets in vivo.

The in vivo response to radiotherapy is not well understood but appears to involve the p53 tumor suppressor protein. We investigated the expression of apoptosis-inducing p53 target genes during gamma-irradiation-induced cell death in p53(+/+) or p53(-/-) mouse tissues using in situ hybridization. Our results reveal striking tissue specificity with distinct regulation of target p53-induced genes in different cells and tissue compartments, as well as variations in dependence on p53 for basal expression. p53-dependent induction of Puma occurred in the splenic white pulp, whereas Noxa and Bid were induced in the red pulp. These patterns correlated with activation of caspase-3 in both compartments. All apoptotic targets of p53 studied here (DR5, Bid, Puma, Noxa) were induced in the jejunum and ileum, which appeared to be the tissues most sensitive to irradiation. We also observed unexpected differences in p53 target gene activation between the transverse and descending colon. Finally, in the liver where irradiation did not lead to caspase-3 activation, we primarily observed p21(WAF1) induction as the major p53-dependent target gene response. Our findings indicate that the selectivity of p53 in transactivation following DNA damage in vivo results in unique tissue and cell type specificity, which may correlate with growth arrest or variable sensitivity to gamma-irradiation.

Farnesyltransferase inhibitors: an overview of the results of preclinical and clinical investigations.

This article presents an overview of preclinical studies and clinical trials of a number of independently derived farnesyltransferase inhibitors (FTIs). Potential targets and biological modes of action of FTIs are discussed, and the results of clinical trials are summarized. The significant efficacy of FTIs as single or combined agents in preclinical studies stands in contrast with only moderate effects in clinical Phase II-III trials. These results reveal a substantial gap in the understanding of the complex activity of FTIs and their interactions with cytotoxic agents. We conclude that the rational combination of FTIs with other therapies, taking into account the biological activities of the individual agents, may improve the clinical results obtained with FTIs.

Preclinical Data on Efficacy of 10 Drug-Radiation Combinations: Evaluations, Concerns, and Recommendations.

BACKGROUND: Clinical testing of new therapeutic interventions requires comprehensive, high-quality preclinical data. Concerns regarding quality of preclinical data have been raised in recent reports. This report examines the data on the interaction of 10 drugs with radiation and provides recommendations for improving the quality, reproducibility, and utility of future studies. The drugs were AZD6244, bortezomib, 17-DMAG, erlotinib, gefitinib, lapatinib, oxaliplatin/Lipoxal, sunitinib (Pfizer, Corporate headquarters, New York, NY), thalidomide, and vorinostat. METHODS: In vitro and in vivo data were tabulated from 125 published papers, including methods, radiation and drug doses, schedules of administration, assays, measures of interaction, presentation and interpretation of data, dosimetry, and conclusions. RESULTS: In many instances, the studies contained inadequate or unclear information that would hamper efforts to replicate or intercompare the studies, and that weakened the evidence for designing and conducting clinical trials. The published reports on these drugs showed mixed results on enhancement of radiation response, except for sunitinib, which was ineffective. CONCLUSIONS: There is a need for improved experimental design, execution, and reporting of preclinical testing of agents that are candidates for clinical use in combination with radiation. A checklist is provided for authors and reviewers to ensure that preclinical studies of drug-radiation combinations meet standards of design, execution, and interpretation, and report necessary information to ensure high quality and reproducibility of studies. Improved design, execution, common measures of enhancement, and consistent interpretation of preclinical studies of drug-radiation interactions will provide rational guidance for prioritizing drugs for clinical radiotherapy trials and for the design of such trials.

Radiation-Therapeutic Agent Clinical Trials: Leveraging Advantages of a National Cancer Institute Programmatic Collaboration.

A number of oncology phase II radiochemotherapy trials with promising results have been conducted late in the overall experimental therapeutic agent development process. Accelerated development and approval of experimental therapeutic agents have stimulated further interest in much earlier radiation-agent studies to increase the likelihood of success in phase III trials. To sustain this interest, more forward-thinking preclinical radiobiology experimental designs are needed to improve discovery of promising radiochemotherapy plus agent combinations for clinical trial testing. These experimental designs should better inform next-step radiation-agent clinical trial dose, schedule, exposure, and therapeutic effect. Recognizing the need for a better strategy to develop preclinical data supporting radiation-agent phase I or II trials, the National Cancer Institute (NCI)-Cancer Therapy Evaluation Program (CTEP) and the NCI-Molecular Radiation Therapeutics Branch of the Radiation Research Program have partnered to promote earlier radiobiology studies of CTEP portfolio agents. In this Seminars in Radiation Oncology article, four key components of this effort are discussed. First, we outline steps for accessing CTEP agents for preclinical testing. Second, we propose radiobiology studies that facilitate transition from preclinical testing to early phase trial activation. Third, we navigate steps that walk through CTEP agent strategic development paths available for radiation-agent testing. Fourth, we highlight a new NCI-sponsored cooperative agreement grant supporting in vitro and in vivo radiation-CTEP agent testing that informs early phase trial designs. Throughout the article, we include contemporary examples of successful radiation-agent development initiatives.

Improving the Predictive Value of Preclinical Studies in Support of Radiotherapy Clinical Trials.

There is an urgent need to improve reproducibility and translatability of preclinical data to fully exploit opportunities for molecular therapeutics involving radiation and radiochemotherapy. For in vitro research, the clonogenic assay remains the current state-of-the-art of preclinical assays, whereas newer moderate and high-throughput assays offer the potential for rapid initial screening. Studies of radiation response modification by molecularly targeted agents can be improved using more physiologic 3D culture models. Elucidating effects on the cancer stem cells (CSC, and CSC-like) and developing biomarkers for defining targets and measuring responses are also important. In vivo studies are necessary to confirm in vitro findings, further define mechanism of action, and address immunomodulation and treatment-induced modification of the microenvironment. Newer in vivo models include genetically engineered and patient-derived xenograft mouse models and spontaneously occurring cancers in domesticated animals. Selection of appropriate endpoints is important for in vivo studies; for example, regrowth delay measures bulk tumor killing, whereas local tumor control assesses effects on CSCs. The reliability of individual assays requires standardization of procedures and cross-laboratory validation. Radiation modifiers must be tested as part of clinical standard of care, which includes radiochemotherapy for most tumors. Radiation models are compatible with but also differ from those used for drug screening. Furthermore, the mechanism of a drug as a chemotherapeutic agent may be different from its interaction with radiation and/or radiochemotherapy. This provides an opportunity to expand the use of molecular-targeted agents. Clin Cancer Res; 22(13); 3138-47. ©2016 AACR.