Identification of granulocyte colony-stimulating factor and interleukin-6 as candidate biomarkers of CBLB502 efficacy as a medical radiation countermeasure.

Given an ever-increasing risk of nuclear and radiological emergencies, there is a critical need for development of medical radiation countermeasures (MRCs) that are safe, easily administered, and effective in preventing and/or mitigating the potentially lethal tissue damage caused by acute high-dose radiation exposure. Because the efficacy of MRCs for this indication cannot be ethically tested in humans, development of such drugs is guided by the Food and Drug Administration's Animal Efficacy Rule. According to this rule, human efficacious doses can be projected from experimentally established animal efficacious doses based on the equivalence of the drug's effects on efficacy biomarkers in the respective species. Therefore, identification of efficacy biomarkers is critically important for drug development under the Animal Efficacy Rule. CBLB502 is a truncated derivative of the Salmonella flagellin protein that acts by triggering Toll-like receptor 5 (TLR5) signaling and is currently under development as a MRC. Here, we report identification of two cytokines, granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6), as candidate biomarkers of CBLB502's radioprotective/mitigative efficacy. Induction of both G-CSF and IL-6 by CBLB502 1) is strictly TLR5-dependent, 2) occurs in a CBLB502 dose-dependent manner within its efficacious dose range in both nonirradiated and irradiated mammals, including nonhuman primates, and 3) is critically important for the ability of CBLB502 to rescue irradiated animals from death. After evaluation of CBLB502 effects on G-CSF and IL-6 levels in humans, these biomarkers will be useful for accurate prediction of human efficacious CBLB502 doses, a key step in the development of this prospective radiation countermeasure.

p53 is a suppressor of inflammatory response in mice.

Chronic inflammation is known to promote cancer, suggesting that negative regulation of inflammation is likely to be tumor suppressive. We found that p53 is a general inhibitor of inflammation that acts as an antagonist of nuclear factor kappaB (NFkappaB). We first observed striking similarities in global gene expression profiles in human prostate cancer cells LNCaP transduced with p53 inhibitory genetic element or treated with TNF, suggesting that p53 inhibits transcription of TNF-inducible genes that are largely regulated by NFkappaB. Consistently, ectopically expressed p53 acts as an inhibitor of transcription of NFkappaB-dependent promoters. Furthermore, suppression of inflammatory response by p53 was observed in vivo in mice by comparing wild-type and p53 null animals at molecular (inhibition of transcription of genes encoding cytokines and chemokines, reducing accumulation of reactive oxygen species and protein oxidation products), cellular (activation of macrophages and neutrophil clearance) and organismal (high levels of metabolic markers of inflammation in tissues of p53-deficient mice and their hypersensitivity to LPS) levels. These observations indicate that p53, acting through suppression of NFkappaB, plays the role of a general "buffer" of innate immune response in vivo that is well consistent with its tumor suppressor function and frequent constitutive activation of NFkappaB in tumors.

Expression of prostate specific antigen (PSA) is negatively regulated by p53.

Although prostate-specific antigen (PSA) is considered a uniquely important tumor marker and is broadly used for early detection of prostate cancer, the molecular mechanisms underlying its elevated expression in tumors have been unknown. By using cDNA microarray gene expression profiling, we found a fourfold increase in the PSA mRNA level in prostatic carcinoma cell line LNCaP, in which the p53 pathway was suppressed by a dominant negative p53 mutant. Consistently, p53 suppression caused a 4-8-fold increase in secretion of PSA protein in culture medium, suggesting that PSA gene expression is under negative control of p53. While wild type p53 strongly repressed, dominant negative p53 mutants stimulated PSA promoter-driven transcription and secretion of PSA in transient transfection experiments. The inhibitory effect of wild type p53 was undetectable in the presence of trichostatin A, suggesting the involvement of histone deacetylation in negative regulation of PSA promoter activity. Thus, PSA is likely to be a tissue specific indicator of transformation-associated p53 suppression in prostate cells. This finding provides a plausible explanation for a frequent increase of PSA levels in advanced prostate cancer.

The Toll-Like Receptor 5 Agonist Entolimod Mitigates Lethal Acute Radiation Syndrome in Non-Human Primates.

There are currently no approved medical radiation countermeasures (MRC) to reduce the lethality of high-dose total body ionizing irradiation expected in nuclear emergencies. An ideal MRC would be effective even when administered well after radiation exposure and would counteract the effects of irradiation on the hematopoietic system and gastrointestinal tract that contribute to its lethality. Entolimod is a Toll-like receptor 5 agonist with demonstrated radioprotective/mitigative activity in rodents and radioprotective activity in non-human primates. Here, we report data from several exploratory studies conducted in lethally irradiated non-human primates (rhesus macaques) treated with a single intramuscular injection of entolimod (in the absence of intensive individualized supportive care) administered in a mitigative regimen, 1-48 hours after irradiation. Following exposure to LD50-70/40 of radiation, injection of efficacious doses of entolimod administered as late as 25 hours thereafter reduced the risk of mortality 2-3-fold, providing a statistically significant (P<0.01) absolute survival advantage of 40-60% compared to vehicle treatment. Similar magnitude of survival improvement was also achieved with drug delivered 48 hours after irradiation. Improved survival was accompanied by predominantly significant (P<0.05) effects of entolimod administration on accelerated morphological recovery of hematopoietic and immune system organs, decreased severity and duration of thrombocytopenia, anemia and neutropenia, and increased clonogenic potential of the bone marrow compared to control irradiated animals. Entolimod treatment also led to reduced apoptosis and accelerated crypt regeneration in the gastrointestinal tract. Together, these data indicate that entolimod is a highly promising potential life-saving treatment for victims of radiation disasters.

Toll-like receptor 5 agonist protects mice from dermatitis and oral mucositis caused by local radiation: implications for head-and-neck cancer radiotherapy.

PURPOSE: Development of mucositis is a frequent side effect of radiotherapy of patients with head-and-neck cancer. We have recently reported that bacterial flagellin, an agonist of Toll-like receptor 5 (TLR5), can protect rodents and primates from acute radiation syndrome caused by total body irradiation. Here we analyzed the radioprotective efficacy of TLR5 agonist under conditions of local, single dose or fractionated radiation treatment. METHODS AND MATERIALS: Mice received either single-dose (10, 15, 20, or 25 Gy) or fractioned irradiation (cumulative dose up to 30 Gy) of the head-and-neck area with or without subcutaneous injection of pharmacologically optimized flagellin, CBLB502, 30 min before irradiation. RESULTS: CBLB502 significantly reduced the severity of dermatitis and mucositis, accelerated tissue recovery, and reduced the extent of radiation induced weight loss in mice after a single dose of 15 or 20 Gy but not 25 Gy of radiation. CBLB502 was also protective from cumulative doses of 25 and 30 Gy delivered in two (10 + 15 Gy) or three (3 × 10 Gy) fractions, respectively. While providing protection to normal epithelia, CBLB502 did not affect the radiosensitivity of syngeneic squamous carcinoma SCCVII grown orthotopically in mice. Use of CBLB502 also elicited a radiation independent growth inhibitory effect upon TLR5-expressing tumors demonstrated in the mouse xenograft model of human lung adenocarcinoma A549. CONCLUSION: CBLB502 combines properties of supportive care (radiotherapy adjuvant) and anticancer agent, both mediated via activation of TLR5 signaling in the normal tissues or the tumor, respectively.

Multiple parameter radiation injury assessment using a nonhuman primate radiation model-biodosimetry applications.

There are urgent needs to establish capability to rapidly assess radiation injury in mass casualty and population monitoring scenarios. This study's objective was to evaluate several currently available biomarkers that can provide early diagnostic triage information after radiation exposure. Hematology and blood chemistry measurements were performed on samples derived from a nonhuman primate (Macaca mulatta; n = 8) total-body irradiation (TBI) model (6.5-Gy Co gamma rays at 0.6 Gy min). The results from this study demonstrate: a) time course for changes in C-reactive protein (CRP) (-2 d to 15 d after TBI); b) time-dependent (-2 d, 1-4 d after TBI) changes in blood cell counts [i.e., lymphocytes decrease to 5-8% of pre-study levels at 1 to 4 d after TBI; ratio of neutrophil to lymphocytes increases by 44 +/- 18 (p = 0.016), 12 +/- 4 (p = 0.001), 8 +/- 2 (p = 0.0020), and 5.0 +/- 2 (p = 0.002) fold at 1, 2, 3, and 4 days after TBI, respectively]; and c) 4.5 +/- 0.8 (p = 0.002)-fold increases in serum amylase activity 1 d after TBI. Plasma CRP levels at 1 d after exposure were 22 +/- 13 (p = 0.0005) (females) and 44 +/- 11 (p = 0.0004) (males)-fold elevated above baseline levels. One hundred percent successful separation of samples from exposed macaques (24 h after TBI) vs. samples from the same macaque taken before irradiation using a discriminant analysis based on four biomarkers (i.e., lymphocytes, neutrophils, ratio of neutrophils to lymphocytes, and serum amylase activity) was demonstrated. These results demonstrate the practical use of multiple parameter biomarkers to enhance the discrimination of exposed vs. non-exposed individuals and justify a follow-on rhesus macaque dose-response study.

An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models.

The toxicity of ionizing radiation is associated with massive apoptosis in radiosensitive organs. Here, we investigate whether a drug that activates a signaling mechanism used by tumor cells to suppress apoptosis can protect healthy cells from the harmful effects of radiation. We studied CBLB502, a polypeptide drug derived from Salmonella flagellin that binds to Toll-like receptor 5 (TLR5) and activates nuclear factor-kappaB signaling. A single injection of CBLB502 before lethal total-body irradiation protected mice from both gastrointestinal and hematopoietic acute radiation syndromes and resulted in improved survival. CBLB502 injected after irradiation also enhanced survival, but at lower radiation doses. It is noteworthy that the drug did not decrease tumor radiosensitivity in mouse models. CBLB502 also showed radioprotective activity in lethally irradiated rhesus monkeys. Thus, TLR5 agonists could potentially improve the therapeutic index of cancer radiotherapy and serve as biological protectants in radiation emergencies.

A systematic search for downstream mediators of tumor suppressor function of p53 reveals a major role of BTG2 in suppression of Ras-induced transformation.

Factors that mediate p53 tumor suppressor activity remain largely unknown. In this study we describe a systematic approach to identify downstream mediators of tumor suppressor function of p53, consisting of global gene expression profiling, focused short hairpin RNA (shRNA) library creation, and functional selection of genetic elements cooperating with oncogenic Ras in cell transformation. This approach is based on our finding that repression of gene expression is a major event, occurring in response to p53 inactivation during transformation and immortalization of primary cells. Functional analysis of the subset of genes universally down-regulated in the cells that lacked functional p53 revealed BTG2 as a major downstream effector of p53-dependent proliferation arrest of mouse and human fibroblasts transduced with oncogenic Ras. shRNA-mediated knockdown of BTG2 cooperates with oncogenic Ras to transform primary mouse fibroblasts containing wild-type transcriptionally active p53. Repression of BTG2 results in up-regulation of cyclins D1 and E1 and phosphorylation of Rb and, in cooperation with other oncogenic elements, induces neoplastic transformation of primary human fibroblasts. BTG2 expression was found to be significantly reduced in a large proportion of human kidney and breast carcinomas, suggesting that BTG2 is a tumor suppressor that links p53 and Rb pathways in human tumorigenesis.