Ilya Gitlin

Core circadian protein CLOCK is a positive regulator of NF-κB–mediated transcription

The circadian clock controls many physiological parameters including immune response to infectious agents, which is mediated by activation of the transcription factor NF-κB. It is widely accepted that circadian regulation is based on periodic changes in gene expression that are triggered by transcriptional activity of the CLOCK/BMAL1 complex. Through the use of a mouse model system we show that daily variations in the intensity of the NF-κB response to a variety of immunomodulators are mediated by core circadian protein CLOCK, which can up-regulate NF-κB–mediated transcription in the absence of BMAL1; moreover, BMAL1 counteracts the CLOCK-dependent increase in the activation of NF-κB–responsive genes. Consistent with its regulatory function, CLOCK is found in protein complexes with the p65 subunit of NF-κB, and its overexpression correlates with an increase in specific phosphorylated and acetylated transcriptionally active forms of p65. In addition, activation of NF-κB in response to immunostimuli in mouse embryonic fibroblasts and primary hepatocytes isolated from Clock-deficient mice is significantly reduced compared with WT cells, whereas Clock-Δ19 mutation, which reduces the transactivation capacity of CLOCK on E-box–containing circadian promoters, has no effect on the ability of CLOCK to up-regulate NF-κB–responsive promoters. These findings establish a molecular link between two essential determinants of the circadian and immune mechanisms, the transcription factors CLOCK and NF-κB, respectively.

Central role of liver in anticancer and radioprotective activities of Toll-like receptor 5 agonist

Significance Toll-like receptor 5 (TLR5) is an innate immunity receptor that specifically recognizes and triggers immune response to bacterial flagellins. In addition to resistance to Salmonella infection, TLR5 agonists protect mammals from radiation and have anticancer effects, including suppression of tumor metastases. Using mouse models, we defined the liver as a major target for TLR5 agonists. Administration of pharmacologically optimized flagellin derivative CBLB502 leads to rapid activation of prosurvival nuclear factor kappa B (NF-κB) and STAT3 pathways in the liver and rescues mice from lethal doses of hepatotoxic Fas-agonistic antibodies. Thus, TLR5 agonists can be considered for treatment and prevention of liver metastasis and hepatoprotective applications. Vertebrate Toll-like receptor 5 (TLR5) recognizes bacterial flagellin proteins and activates innate immune responses to motile bacteria. In addition, activation of TLR5 signaling can inhibit growth of TLR5-expressing tumors and protect normal tissues from radiation and ischemia-reperfusion injuries. To understand the mechanisms behind these phenomena at the organismal level, we assessed nuclear factor kappa B (NF-κB) activation (indicative of TLR5 signaling) in tissues and cells of mice treated with CBLB502, a pharmacologically optimized flagellin derivative. This identified the liver and gastrointestinal tract as primary CBLB502 target organs. In particular, liver hepatocytes were the main cell type directly and specifically responding to systemic administration of CBLB502 but not to that of the TLR4 agonist LPS. To assess CBLB502 impact on other pathways, we created multireporter mice with hepatocytes transduced in vivo with reporters for 46 inducible transcription factor families and found that along with NF-κB, CBLB502 strongly activated STAT3-, phenobarbital-responsive enhancer module (PREM), and activator protein 1 (AP-1–) -driven pathways. Livers of CBLB502-treated mice displayed induction of numerous immunomodulatory factors and massive recruitment of various types of immune cells. This led to inhibition of growth of liver metastases of multiple tumors regardless of their TLR5 status. The changed liver microenvironment was not, however, hepatotoxic, because CBLB502 induced resistance to Fas-mediated apoptosis in normal liver cells. Temporary occlusion of liver blood circulation prevented CBLB502 from protecting hematopoietic progenitors in lethally irradiated mice, indicating involvement of a factor secreted by responding liver cells. These results define the liver as the key mediator of TLR5-dependent effects in vivo and suggest clinical applications for TLR5 agonists as hepatoprotective and antimetastatic agents.

Aging of mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-galpH6), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-galpH6-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-galpH6-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-galpH6-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-galpH6-positive cells and reconsideration of potential cellular target for anti-aging treatment.

p16(Ink4a) and senescence-associated β-galactosidase can be induced in macrophages as part of a reversible response to physiological stimuli

Constitutive p16Ink4a expression, along with senescence-associated β-galactosidase (SAβG), are commonly accepted biomarkers of senescent cells (SCs). Recent reports attributed improvement of the healthspan of aged mice following p16Ink4a-positive cell killing to the eradication of accumulated SCs. However, detection of p16Ink4a/SAβG-positive macrophages in the adipose tissue of old mice and in the peritoneal cavity of young animals following injection of alginate-encapsulated SCs has raised concerns about the exclusivity of these markers for SCs. Here we report that expression of p16Ink4a and SAβG in macrophages is acquired as part of a physiological response to immune stimuli rather than through senescence, consistent with reports that p16Ink4a plays a role in macrophage polarization and response. Unlike SCs, p16Ink4a/SAβG-positive macrophages can be induced in p53-null mice. Macrophages, but not mesenchymal SCs, lose both markers in response to M1- [LPS, IFN-α, Poly(I:C)] and increase their expression in response to M2-inducing stimuli (IL-4, IL-13). Moreover, interferon-inducing agent Poly(I:C) dramatically reduced p16Ink4a expression in vivo in our alginate bead model and in the adipose tissue of aged mice. These observations suggest that the antiaging effects following eradication of p16Ink4a-positive cells may not be solely attributed to SCs but also to non-senescent p16Ink4a/SAβG-positive macrophages.

Physiological frailty index (PFI): quantitative in-life estimate of individual biological age in mice

The development of healthspan-extending pharmaceuticals requires quantitative estimation of age-related progressive physiological decline. In humans, individual health status can be quantitatively assessed by means of a frailty index (FI), a parameter which reflects the scale of accumulation of age-related deficits. However, adaptation of this methodology to animal models is a challenging task since it includes multiple subjective parameters. Here we report a development of a quantitative non-invasive procedure to estimate biological age of an individual animal by creating physiological frailty index (PFI). We demonstrated the dynamics of PFI increase during chronological aging of male and female NIH Swiss mice. We also demonstrated acceleration of growth of PFI in animals placed on a high fat diet, reflecting aging acceleration by obesity and provide a tool for its quantitative assessment. Additionally, we showed that PFI could reveal anti-aging effect of mTOR inhibitor rapatar (bioavailable formulation of rapamycin) prior to registration of its effects on longevity. PFI revealed substantial sex-related differences in normal chronological aging and in the efficacy of detrimental (high fat diet) or beneficial (rapatar) aging modulatory factors. Together, these data introduce PFI as a reliable, non-invasive, quantitative tool suitable for testing potential anti-aging pharmaceuticals in pre-clinical studies.

TRAIN (Transcription of Repeats Activates INterferon) in response to chromatin destabilization induced by small molecules in mammalian cells

Cellular responses to the loss of genomic stability are well-established, while how mammalian cells respond to chromatin destabilization is largely unknown. We previously found that DNA demethylation on p53-deficient background leads to transcription of repetitive heterochromatin elements, followed by an interferon response, a phenomenon we named TRAIN (Transcription of Repeats Activates INterferon). Here, we report that curaxin, an anticancer small molecule, destabilizing nucleosomes via disruption of histone/DNA interactions, also induces TRAIN. Furthermore, curaxin inhibits oncogene-induced transformation and tumor growth in mice in an interferon-dependent manner, suggesting that anticancer activity of curaxin, previously attributed to p53-activation and NF-kappaB-inhibition, may also involve induction of interferon response to epigenetic derepression of the cellular ‘repeatome’. Moreover, we observed that another type of drugs decondensing chromatin, HDAC inhibitor, also induces TRAIN. Thus, we proposed that TRAIN may be one of the mechanisms ensuring epigenetic integrity of mammalian cells via elimination of cells with desilenced chromatin.

Inhibition of the aryl hydrocarbon receptor/polyamine biosynthesis axis suppresses multiple myeloma

Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent.

PO-459 Systemic anti-inflammatory effect of a new anticancer cyclic peptide ALOS4

Introduction Chronic inflammation is the primary pathological element of cancer development. Here we demonstrate a synthetic cyclic peptide ALOS4 capable of suppressing tumour growth following systemic administration and possessing anti-inflammatory properties. ALOS4 was initially isolated by phage display for its ability to bind αvβ3 integrin, possessing no functional activity of RGD peptides and characterised by tumour suppressor activity in preclinical xenograft and syngeneic models of melanoma. Importantly, ALOS4 displayed no signs of toxicity in treated animals even at the highest doses, which significantly exceeded optimal efficacious doses. In this study, we aimed to identify the mechanisms underlying the profound antitumor activity of this safe peptide. Material and methods We utilised a large panel of cell-based repeater assays designed to detect modulators of a variety of signalling pathways including p53-, NF-kB-, hypoxia, heat shock, irradiation and other types of stress responses. We also tested the effects of ALOS4 on tumour cell growth, adhesion, migration, clonogenicity, morphology and other in vitro properties. Results and discussions ALOS4 showed no detectable activity in most of these assays with some exceptions: first, ALOS4 treatment prevented weight lost in cancer bearing mice and second, ALOS4 suppressed the ability of treated cells to induce interferon type I signalling in response to double stranded RNA mimics (Poly I:C). Based on this observation, as well as negative in vitro cell-based assay results, we hypothesised that the antitumor effect of ALOS4 may be mediated by a systemic anti-inflammatory effect rather than by a direct effect on tumour cells. Consistent with this hypothesis, ALOS4 treatment dramatically modulated the abundance and content of the population of immunocytes infiltrating subcutaneously growing melanomas in mice. Conclusion These results suggest that ALOS4 can represent an anticancer agent with a new mechanism of activity that targets the tumor-supporting interferon-driven mechanism of tumor-host interaction and improves the overall condition of the mouse as a whole.

Abstract 4398: Liver is a major primary target for the Toll-like receptor-5 agonist CBLB502 providing radioprotective, antimicrobial and antitumor responses

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL We have previously shown that activation of Toll-like receptor 5 (TLR5) by bacterial flagellin and by pharmacologically optimized flagellin derivative CBLB502 protects mice and monkeys from lethal radiation injury to the hematopoietic and gastrointestinal systems. Here, we report that the liver is a major primary CBLB502 target organ in mice, with hepatocytes specifically showing rapid and strong NF-kB and STAT3 activation. Livers from CBLB502-treated mice showed up-regulation of numerous downstream genes encoding intracellular and secreted proteins with anti-apoptotic, anti-microbial and immunomodulatory activities. Unlike the TLR4 agonist LPS, the liver response to TLR5 agonists appears to be direct and does not involve high levels of proinflammatory cytokines. Thus, while LPS is toxic, TLR5 agonists have strong clinical potential. The importance of liver for CBLB502 radioprotective activity was confirmed by temporary occlusion of liver blood circulation which completely abrogated the protective effect of CBLB502 on hematopoietic precursor cells in irradiated mice. CBLB502 also protected liver tissue itself, increasing mouse resistance to lethal Salmonella tiphymurium liver infection and to hepatotoxic Fas agonistic antibodies. By testing CBLB502 in combination with radiation treatment of experimental mouse tumors in vivo, we demonstrated that its tissue protection properties are limited to normal tissues with no tumor protection detected in any of numerous mouse tumor models. Moreover, direct antitumor effects of CBLB502 treatment was observed in several tumor models. Comparison of the effect of CBLB502 on in vivo growth of isogenic pairs of tumor cell lines differing in their TLR5 status showed that the antitumor effect of the TLR5 agonist is TLR5 dependent and is associated with tumor infiltration by immunocytes, presumably attracted following activation of TLR5 signaling in the tumor cells. Remarkably, CBLB502 caused an immunotherapeutic effect in TLR5-negative tumors (CT26 colon adenocarcinoma and A20 lymphoma) growing as experimental liver metastases. Based on these results, we project clinical applications of CBLB502 as an anticancer immunotherapeutic drug against liver metastases independently on TLR5 status and TLR5-expressing tumors in other locations as well as supporting care drug to reduce adverse hepatotoxicity from radiation and chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4398. doi:1538-7445.AM2012-4398

Abstract A63: TLR5 agonist CBLB502 protects mice from Fas-mediated liver damage without protecting tumors

We have previously shown the strong radioprotective properties of the Toll-like receptor 5 (TLR5) agonist flagellin and its derivative CBLB502 in both mouse and non-human primate models (Burdelya et al., 2008, Science 320: 226–230). Because no protection has been observed in any in vitro cell model regardless of the TLR5 status of the cells, we hypothesized that the radioprotective effects of the TLR5 agonist are indirect and mediated by factors produced by primary cells responding to flagellin in the organism. Using a combination of tools, including NF-kB-responsive reporter mice and immunohistochemical determination of tissues responding to CBLB502 by NF-kB activation, we defined liver hepatocytes as the primary pharmacological targets of flagellin. Surgical exclusion of the liver from the blood circulation completely abolished radioprotection of the bone marrow hematopoietic progenitor cells by CBLB502 suggesting that the liver acts as a mediator of at least some of the effects of the TLR5 agonist. Since the induction of NF-kB is known to activate multiple pro-survival mechanisms, we further hypothesized that administration of the TLR5 agonist can result in liver resistance to otherwise toxic conditions. Mice treated with Fas agonistic antibodies were employed as a model of acute hepatotoxicity. Injection of CBLB502 showed a strong preventive effect against Fas-mediated injury rescuing all of the injected mice in contrast to complete lethality in the control groups. The ability of CBLB502 to protect the liver was also demonstrated via reduced levels of liver enzymes in the peripheral blood and caspase activation in liver extracts of mice treated with Fas agonistic antibodies. Using two syngeneic tumor models, colon adenocarcinoma CT26 and lymphoma A20, growing as liver metastases in mice, we found that CBLB502 injections not only prevented liver damage, but also suppressed tumor appearance and growth when injected with or without anti-Fas antibodies. Thus, TLR5 agonist has potential to become a new agent for liver protection and suppression of liver metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A63.