Neuroimmune mechanisms of behavioral alterations in a syngeneic murine model of human papilloma virus-related head and neck cancer.

Patients with cancer often experience a high symptom burden prior to the start of treatment. As disease- and treatment-related neurotoxicities appear to be additive, targeting disease-related symptoms may attenuate overall symptom burden for cancer patients and improve the tolerability of treatment. It has been hypothesized that disease-related symptoms are a consequence of tumor-induced inflammation. We tested this hypothesis using a syngeneic heterotopic murine model of human papilloma virus (HPV)-related head and neck cancer. This model has the advantage of being mildly aggressive and not causing cachexia or weight loss. We previously showed that this tumor leads to increased IL-6, IL-1ß, and TNF-α expression in the liver and increased IL-1ß expression in the brain. The current study confirmed these features and demonstrated that the tumor itself exhibits high inflammatory cytokine expression (e.g., IL-6, IL-1ß, and TNF-α) compared to healthy tissue. While there is a clear relationship between cytokine levels and behavioral deficits in this model, the behavioral changes are surprisingly mild. Therefore, we sought to confirm the relationship between behavior and inflammation by amplifying the effect using a low dose of lipopolysaccharide (LPS, 0.1mg/kg). In tumor-bearing mice LPS induced deficits in nest building, tail suspension, and locomotor activity approximately 24h after LPS. However, these mice did not display an exacerbation of LPS-induced weight loss, anorexia, or anhedonia. Further, while heightened serum IL-6 was observed there was minimal priming of liver or brain cytokine expression. Next we sought to inhibit tumor-induced burrowing deficits by reducing inflammation using minocycline. Minocycline (∼50mg/kg/day in drinking water) was able to attenuate tumor-induced inflammation and burrowing deficits. These data provide evidence in favor of an inflammatory-like mechanism for the behavioral alterations associated with tumor growth in a syngeneic murine model of HPV-related head and neck cancer. However, the inflammatory state and behavioral changes induced by this tumor clearly differ from other forms of inflammation-induced sickness behavior.

The Limitations of Model-Based Experimental Design and Parameter Estimation in Sloppy Systems.

We explore the relationship among experimental design, parameter estimation, and systematic error in sloppy models. We show that the approximate nature of mathematical models poses challenges for experimental design in sloppy models. In many models of complex biological processes it is unknown what are the relevant physical mechanisms that must be included to explain system behaviors. As a consequence, models are often overly complex, with many practically unidentifiable parameters. Furthermore, which mechanisms are relevant/irrelevant vary among experiments. By selecting complementary experiments, experimental design may inadvertently make details that were ommitted from the model become relevant. When this occurs, the model will have a large systematic error and fail to give a good fit to the data. We use a simple hyper-model of model error to quantify a model's discrepancy and apply it to two models of complex biological processes (EGFR signaling and DNA repair) with optimally selected experiments. We find that although parameters may be accurately estimated, the discrepancy in the model renders it less predictive than it was in the sloppy regime where systematic error is small. We introduce the concept of a sloppy system-a sequence of models of increasing complexity that become sloppy in the limit of microscopic accuracy. We explore the limits of accurate parameter estimation in sloppy systems and argue that identifying underlying mechanisms controlling system behavior is better approached by considering a hierarchy of models of varying detail rather than focusing on parameter estimation in a single model.

The Withers archive: Online availability of rod Wither's data.

The contents of the lab notebooks of H.R. Withers have been digitized and stored as 23 excel files, a total of approximately 45 megabytes. A procedure is described whereby those interested may gain access to the data.

Sickness behavior induced by cisplatin chemotherapy and radiotherapy in a murine head and neck cancer model is associated with altered mitochondrial gene expression.

The present study was undertaken to explore the possible mechanisms of the behavioral alterations that develop in response to cancer and to cancer therapy. For this purpose we used a syngeneic heterotopic mouse model of human papilloma virus (HPV)-related head and neck cancer in which cancer therapy is curative. Mice implanted or not with HPV+ tumor cells were exposed to sham treatment or a regimen of cisplatin and radiotherapy (chemoradiation). Sickness was measured by body weight loss and reduced food intake. Motivation was measured by burrowing, a highly prevalent species specific behavior. Tumor-bearing mice showed a gradual decrease in burrowing over time and increased brain and liver inflammatory cytokine mRNA expression by 28 days post tumor implantation. Chemoradiation administered to healthy mice resulted in a mild decrease in burrowing, body weight, and food intake. Chemoradiation in tumor-bearing mice decreased tumor growth and abrogated liver and brain inflammation, but failed to attenuate burrowing deficits. PCR array analysis of selected hypoxia and mitochondrial genes revealed that both the tumor and chemoradiation altered the expression of genes involved in mitochondrial energy metabolism within the liver and brain and increased expression of genes related to HIF-1α signaling within the brain. The most prominent changes in brain mitochondrial genes were noted in tumor-bearing mice treated with chemoradiation. These findings indicate that targeting mitochondrial dysfunction following cancer and cancer therapy may be a strategy for prevention of cancer-related symptoms.

A high content clonogenic survival drug screen identifies mek inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer.

INTRODUCTION: Traditional clonogenic survival and high throughput colorimetric assays are inadequate as drug screens to identify novel radiation sensitizers. We developed a method that we call the high content clonogenic survival assay (HCSA) that will allow screening of drug libraries to identify candidate radiation sensitizers. METHODS: Drug screen using HCSA was done in 96 well plates. After drug treatment, irradiation, and incubation, colonies were stained with crystal violet and imaged on the INCell 6000 (GE Health). Colonies achieving 50 or more cells were enumerated using the INCell Developer image analysis software. A proof-of-principle screen was done on the KRAS mutant lung cancer cell line H460 and a Custom Clinical Collection (146 compounds). RESULTS: Multiple drugs of the same class were found to be radiation sensitizers and levels of potency seemed to reflect the clinical relevance of these drugs. For instance, several PARP inhibitors were identified as good radiation sensitizers in the HCSA screen. However, there were also a few PARP inhibitors not found to be sensitizing that have either not made it into clinical development, or in the case of BSI-201, was proven to not even be a PARP inhibitor. We discovered that inhibitors of pathways downstream of activated mutant KRAS (PI3K, AKT, mTOR, and MEK1/2) sensitized H460 cells to radiation. Furthermore, the potent MEK1/2 inhibitor tramenitib selectively enhanced radiation effects in KRAS mutant but not wild-type lung cancer cells. CONCLUSIONS: Drug screening for novel radiation sensitizers is feasible using the HCSA approach. This is an enabling technology that will help accelerate the discovery of novel radiosensitizers for clinical testing.

Use of the LQ model with large fraction sizes results in underestimation of isoeffect doses.

PURPOSE: To test the appropriateness of the linear-quadratic (LQ) model to describe survival of jejunal crypt clonogens after split doses with variable (small 1-6 Gy, large 8-13 Gy) first dose, as a model of its appropriateness for both small and large fraction sizes. METHODS: C3Hf/KamLaw mice were exposed to whole body irradiation using 300 kVp X-rays at a dose rate of 1.84 Gy/min, and the number of viable jejunal crypts was determined using the microcolony assay. 14 Gy total dose was split into unequal first and second fractions separated by 4 h. Data were analyzed using the LQ model, the lethal potentially lethal (LPL) model, and a repair-saturation (RS) model. RESULTS: Cell kill was greater in the group receiving the larger fraction first, creating an asymmetry in the plot of survival vs size of first dose, as opposed to the prediction of the LQ model of a symmetric response. There was a significant difference in the estimated ßs (higher ß after larger first doses), but no significant difference in the αs, when large doses were given first vs small doses first. This difference results in underestimation (based on present data by approximately 8%) of isoeffect doses using LQ model parameters based on small fraction sizes. While the LPL model also predicted a symmetric response inconsistent with the data, the RS model results were consistent with the observed asymmetry. CONCLUSION: The LQ model underestimates doses for isoeffective crypt-cell survival with large fraction sizes (in the present setting, >9 Gy).

Mitigation and treatment of radiation-induced thoracic injury with a cyclooxygenase-2 inhibitor, celecoxib.

PURPOSE: To test whether a cyclooxygenase-2 inhibitor (celecoxib) could reduce mortality resulting from radiation-induced pneumonitis. METHODS AND MATERIALS: Celecoxib was given to mice twice daily for 40 consecutive days starting on the day of local thoracic irradiation (LTI) or 40 or 80 days later. C3Hf/KamLaw mice were observed for morbidity, and time to death was determined. Results were analyzed using the Cox proportional hazards model. RESULTS: Timing of celecoxib relative to LTI determined efficacy. A significant reduction in time to death was achieved only when celecoxib was started 80 days after LTI, corresponding to the time when pneumonitis is expressed. For these mice the reduction in mortality was quantified as a hazard ratio for mortality of treated vs untreated of 0.36 (95% confidence interval [CI] 0.24-0.53), thus significantly less than 1.0. Correspondingly, the median lethal dose for treated mice (12.9 Gy; 95% CI 12.55-13.25 Gy) was significantly (P=.026) higher than for untreated mice (12.4 Gy; 95% CI 12.2-12.65 Gy). CONCLUSIONS: Celecoxib significantly reduced lung toxicity when administered months after LTI when the deleterious effects of radiation were expressed. The schedule-dependent reduction in fatal pneumonitis suggests that celecoxib could be clinically useful by reintroduction of treatment months after completion of radiation therapy. These findings may be important for designing clinical trials using cyclooxygenase-2 inhibitors to treat radiation-induced lung toxicity as a complement to concurrent radiation therapy of lung cancers.

CpG plus radiotherapy: a review of preclinical works leading to clinical trial.

Studies performed three decades ago in our laboratory supported the hypothesis that radiation efficacy may be augmented by bacterial extracts that stimulate non-specific systemic antitumor immune responses. Application to the clinic was halted by unacceptable side effects and toxicities resulting from exposure to whole bacterial pathogens. Later scientific advances demonstrated that DNA isolated from bacteria was immunostimulatory and could be reproduced with synthetic oligodeoxynucleotides (ODNs), thus fueling the transition from bugs to drugs. Unmethylated CpG motifs within bacterial DNA induce activation of Toll-like receptor 9 and subsequently activate antigen-specific cellular immune responses. CpG ODNs have demonstrated favorable toxicity profiles in phase I clinical trials. We showed that this potent immunoadjuvant can be used in combination with radiation therapy to enhance local and systemic responses of several murine tumors. Studies demonstrated that enhanced tumor response is mediated in part by the host immune system. Antitumor efficacy was diminished in immunocompromised mice. Animals cured by combination of radiation and CpG ODN were resistant to subsequent tumor rechallenge. This body of work contributes to our understanding of the dynamic interplay between tumor irradiation and the host immune system and may facilitate translation to clinical trials.

MK-4827, a PARP-1/-2 inhibitor, strongly enhances response of human lung and breast cancer xenografts to radiation.

The poly-(ADP-ribose) polymerase (PARP) inhibitor, MK-4827, is a novel potent, orally bioavailable PARP-1 and PARP-2 inhibitor currently in phase I clinical trials for cancer treatment. No preclinical data currently exist on the combination of MK-4827 with radiotherapy. The current study examined combined treatment efficacy of MK-4827 and fractionated radiotherapy using a variety of human tumor xenografts of differing p53 status: Calu-6 (p53 null), A549 (p53 wild-type [wt]) and H-460 (p53 wt) lung cancers and triple negative MDA-MB-231 human breast carcinoma. To mimic clinical application of radiotherapy, fractionated radiation (2 Gy per fraction) schedules given once or twice daily for 1 to 2 weeks combined with MK-4827, 50 mg/kg once daily or 25 mg/kg twice daily, were used. MK-4827 was found to be highly and similarly effective in both radiation schedules but maximum radiation enhancement was observed when MK-4827 was given at a dose of 50 mg/kg once daily (EF = 2.2). MK-4827 radiosensitized all four tumors studied regardless of their p53 status. MK-4827 reduced PAR levels in tumors by 1 h after administration which persisted for up to 24 h. This long period of PARP inhibition potentially adds to the flexibility of design of future clinical trials. Thus, MK-4827 shows high potential to improve the efficacy of radiotherapy.

Animal models for medical countermeasures to radiation exposure.

Since September 11, 2001, there has been the recognition of a plausible threat from acts of terrorism, including radiological or nuclear attacks. A network of Centers for Medical Countermeasures against Radiation (CMCRs) has been established across the U.S.; one of the missions of this network is to identify and develop mitigating agents that can be used to treat the civilian population after a radiological event. The development of such agents requires comparison of data from many sources and accumulation of information consistent with the "Animal Rule" from the Food and Drug Administration (FDA). Given the necessity for a consensus on appropriate animal model use across the network to allow for comparative studies to be performed across institutions, and to identify pivotal studies and facilitate FDA approval, in early 2008, investigators from each of the CMCRs organized and met for an Animal Models Workshop. Working groups deliberated and discussed the wide range of animal models available for assessing agent efficacy in a number of relevant tissues and organs, including the immune and hematopoietic systems, gastrointestinal tract, lung, kidney and skin. Discussions covered the most appropriate species and strains available as well as other factors that may affect differential findings between groups and institutions. This report provides the workshop findings.

Importance of maintenance therapy in C225-induced enhancement of tumor control by fractionated radiation.

PURPOSE: C225 strongly enhances tumor radioresponse when given concurrently with radiotherapy. We investigated whether additional therapeutic benefit could be achieved by continuing maintenance treatment with C225 after the completion of fractionated radiotherapy. METHODS AND MATERIALS: A431 xenografts were treated with local irradiation or combined with C225 by two different schedules: (1) 6 h before the first dose of irradiation and at 3-day intervals for a total of 3 doses during the 7-day fractionated radiotherapy, or (2) 6 doses of C225 given both during radiotherapy and continuing for 3 additional doses after radiotherapy. Tumor cure was assessed by the radiation dose yielding local tumor control in 50% of animals (TCD50), and time to recurrence was also determined. RESULTS: Both treatment schedules increased radiocurability as evidenced by reductions in TCD50, but the effect was greater when C225 was given both during and after radiotherapy. C225 reduced the TCD50 of 83.1 (73.2-124.8) Gy by radiation only to 46.2 (39.1-57.5) Gy when given during radiotherapy and to 30.8 (22.2-38.0) Gy when given during and after radiotherapy. Dose modification factors were 1.8 when C225 was given during radiotherapy and 2.7 when given both during and after radiotherapy. C225 was also effective in delaying the onset of tumor recurrences, and was more effective when given as both concurrent and maintenance therapy. CONCLUSIONS: Data showed that C225 strongly enhanced the curative effect of fractionated radiation, and its effect was greater if administration was extended beyond the end of radiotherapy. This important finding may influence future designs of clinical trials combining anti-EGFR (anti-epidermal growth factor receptor) agents with radiotherapy.

Flavopiridol increases therapeutic ratio of radiotherapy by preferentially enhancing tumor radioresponse.

PURPOSE: Recently we reported that inhibition of cyclin-dependent kinases (cdks) by flavopiridol enhanced the radiation response of murine ovarian carcinoma cells in culture. The purpose of this investigation was to extend these studies to in vivo tumor models and test whether flavopiridol increases the therapeutic ratio of radiotherapy. METHODS AND MATERIALS: Three transplantable syngeneic mouse tumors were used: mammary carcinoma (MCa-29), ovarian carcinoma (OCa-I), and a lymphoma (Ly-TH). Tumor treatment endpoints included growth delay, cure, and spontaneous lung metastases (OCa-I tumor). The normal tissue endpoint was survival of jejunal crypt cells quantified microscopically. A range of flavopiridol doses from 0.625 to 5.0 mg/kg were given systemically once or twice daily over 5, 10, or 20 days. Combined therapy flavopiridol treatments were initiated either several days before or shortly after the start of single dose or daily fractionated radiotherapy. RESULTS: The major findings of this study are that all three tumors treated with flavopiridol alone responded by tumor growth delay. Two of the tumors (MCa-29 and Ly-TH) responded in a schedule-dependent manner with larger radiation enhancement factors when flavopiridol treatment was started a few hours after irradiation (radioenhancement factors [EF] Ly-TH = 2.04, EF MCa-29 = 1.50 for single dose irradiation). When combined with fractionated irradiation (2.6 Gy daily for 10 or 20 days), flavopiridol enhanced the response of the MCa-29 tumor by a factor of 1.25-1.46. A fractional radiation dose of 6 Gy in combination with flavopiridol produced a 62.5% cure rate compared with 25% tumor cure for radiation alone. A novel finding of this study was the demonstration of antimetastatic activity of flavopiridol in addition to its effect on the local primary tumor. Both the incidence and absolute number of lung metastasis were reduced when flavopiridol followed surgical removal of the large (10 mm) primary leg tumor. The normal jejunum treated with flavopiridol and radiation responded in a schedule independent manner and the degree of radioenhancement (EF, 1.05-1.06) was much less than for any of the tumors studied. CONCLUSIONS: Therapeutic gain was achieved when flavopiridol treatment was initiated either before or after the start of radiotherapy. Flavopiridol shows promising clinical potential administered alone or in combination with other cytotoxic agents, including both chemotherapy and radiotherapy.

Radiosensitization of human and rodent cell lines by INO-1001, a novel inhibitor of poly(ADP-ribose) polymerase.

Inhibition of poly(ADP-ribose) polymerase (PARP) by a novel, potent inhibitor, INO-1001, was examined in two rodent and one human fibroblast cell lines, after single and fractionated radiation treatments. Since PARP plays a role in the early events following DNA damage and influences the effectiveness of DNA repair, its inhibition has been proposed to constitute a drug target for the development of novel radiosensitizers. We found that INO-1001 effectively inhibited PARP activity at non-cytotoxic concentrations. Combination treatment of 10 microM INO-1001 and a single dose of radiation resulted in significant radiosensitization of all three cells lines (enhancement ratios 1.4-1.6). This radioenhancement was even greater when the drug and radiation were given as fractionated treatments (enhancement ratio 8.0). Apoptosis (as evaluated by TUNEL staining) was not enhanced by the treatments, suggesting that inhibiting PARP enzyme activity by INO-1001 enhanced radiation-induced cell killing by interfering with DNA repair mechanisms, resulting in necrotic cell death. INO-1001 therefore, appears to have potential as a potent enhancer of radiation sensitivity, without any intrinsic cytotoxicity from the drug alone.

Flavopiridol, a cyclin-dependent kinase inhibitor, enhances radiosensitivity of ovarian carcinoma cells.

Flavopiridol, a cyclin-dependent kinase (cdk) inhibitor, can cause cell cycle arrest, induce apoptosis in cancer cells, and inhibit tumor cell growth in vivo. The present study investigated the in vitro radiosensitizing effect of flavopiridol and the underlying molecular mechanisms in a murine ovarian cancer cell line, OCA-I. Flavopiridol inhibited cell growth in a dose-dependent manner and enhanced cell radiosensitivity assessed by the clonogenic cell survival assay. A flavopiridol dose of 300 nM, given for 1 day, enhanced radiosensitivity by a factor of 2.1. Clonogenic cell survival after split-dose radiation showed that flavopiridol inhibited repair from radiation damage. In addition, flavopiridol treatment (300 nM, 1 day) resulted in decreased levels of Ku70 and Ku86 proteins that play a role in DNA repair processes, suggesting that DNA repair processes may have been disrupted by this agent. Flow cytometry analysis showed that flavopiridol (300 nM, 1 day) accumulated the cells in G(1) and G(2) phases, with a significant reduction in the S phase component. This cell cycle redistribution is likely another mechanism underlying flavopiridol-induced cell radiosensitivity. Flavopiridol down-regulated cyclin D1 and cyclin E protein levels and also inhibited phosphorylation of retinoblastoma protein, which is inconsistent with the observed cell cycle arrest. Among the cdks tested, cdk-9, the catalytic subunit of positive transcription elongation factor b, was significantly down-regulated by flavopiridol, suggesting that flavopiridol may modulate cellular transcription processes. Furthermore, flavopiridol on its own induced apoptosis in the OCA-I cells, whereas in combination with radiation, exerted no additional increase in apoptosis. Taken together, our data show that flavopiridol strongly augmented the response of ovarian carcinoma cells to radiation and that the underlying mechanisms included inhibition of sublethal DNA damage repair and cell cycle redistribution. At the molecular level, transcriptional regulation by flavopiridol may have been involved.