Katerina I. Leonova

p53 cooperates with DNA methylation and a suicidal interferon response to maintain epigenetic silencing of repeats and noncoding RNAs

Large parts of mammalian genomes are transcriptionally inactive and enriched with various classes of interspersed and tandem repeats. Here we show that the tumor suppressor protein p53 cooperates with DNA methylation to maintain silencing of a large portion of the mouse genome. Massive transcription of major classes of short, interspersed nuclear elements (SINEs) B1 and B2, both strands of near-centromeric satellite DNAs consisting of tandem repeats, and multiple species of noncoding RNAs was observed in p53-deficient but not in p53 wild-type mouse fibroblasts treated with the DNA demethylating agent 5-aza-2’-deoxycytidine. The abundance of these transcripts exceeded the level of β-actin mRNA by more than 150-fold. Accumulation of these transcripts, which are capable of forming double-stranded RNA (dsRNA), was accompanied by a strong, endogenous, apoptosis-inducing type I IFN response. This phenomenon, which we named “TRAIN” (for “transcription of repeats activates interferon”), was observed in spontaneous tumors in two models of cancer-prone mice, presumably reflecting naturally occurring DNA hypomethylation and p53 inactivation in cancer. These observations suggest that p53 and IFN cooperate to prevent accumulation of cells with activated repeats and provide a plausible explanation for the deregulation of IFN function frequently seen in tumors. Overall, this work reveals roles for p53 and IFN that are key for genetic stability and therefore relevant to both tumorigenesis and the evolution of species.

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.

Nrf2 Amplifies Oxidative Stress via Induction of Klf9

Reactive oxygen species (ROS) activate NF-E2-related transcription factor 2 (Nrf2), a key transcriptional regulator driving antioxidant gene expression and protection from oxidant injury. Here, we report that in response to elevation of intracellular ROS above a critical threshold, Nrf2 stimulates expression of transcription Kruppel-like factor 9 (Klf9), resulting in further Klf9-dependent increases in ROS and subsequent cell death. We demonstrated that Klf9 independently causes increased ROS levels in various types of cultured cells and in mouse tissues and is required for pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Mechanistically, Klf9 binds to the promoters and alters the expression of several genes involved in the metabolism of ROS, including suppression of thioredoxin reductase 2, an enzyme participating in ROS clearance. Our data reveal an Nrf2-dependent feedforward regulation of ROS and identify Klf9 as a ubiquitous regulator of oxidative stress and lung injury.

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.

A small molecule inhibitor of p53 stimulates amplification of hematopoietic stem cells but does not promote tumor development in mice

It has been shown that genetic inhibition of p53 leads to enhanced proliferation of hematopoietic stem cells (HSCs). This could, in theory, contribute to the increased frequency of tumor development observed in p53-deficient mice and humans. In our previous work, we identified chemical p53 inhibitors (PFTs) that suppress the transactivation function of p53 and protect cultured cells and mice from death induced by gamma irradiation (IR). Here we found that when applied to bone marrow cells in vitro or injected into mice, PFTb impeded IR-induced reduction of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) population sizes. In addition, we showed that PFTb stimulated HSC and HPC proliferation in the absence of IR in vitro and in vivo and mobilized HSCs to the peripheral blood. Importantly, however, PFTb treatment did not affect the timing or frequency of tumor development in irradiated p53 heterozygous mice used as a model for determination of carcinogenicity. Thus, although PFTb administration led to increased numbers of HSCs and HPCs, it was not carcinogenic in mice. These findings suggest that chemical p53 inhibitors may be clinically useful as safe and effective stimulators of hematopoiesis.

Distinguishing the immunostimulatory properties of noncoding RNAs expressed in cancer cells

Significance Using an approach derived from statistical physics, we quantify transcriptome-wide motif usage in human and murine noncoding RNAs (ncRNAs), determining that most have motif usage consistent with the coding genome. However, an outlier subset of tumor-associated ncRNAs comprises repetitive elements whose motif usage patterns are more typically associated with the genomes of inflammatory pathogens. We demonstrate that a key subset of these elements directly activates the cellular innate immune response. We propose that the innate response in tumors partially originates from direct interaction of immunogenic ncRNAs preferentially expressed in cancer cells with innate pattern recognition receptors. Recent studies have demonstrated abundant transcription of a set of noncoding RNAs (ncRNAs) preferentially within tumors as opposed to normal tissue. Using an approach from statistical physics, we quantify global transcriptome-wide motif use for the first time, to our knowledge, in human and murine ncRNAs, determining that most have motif use consistent with the coding genome. However, an outlier subset of tumor-associated ncRNAs, typically of recent evolutionary origin, has motif use that is often indicative of pathogen-associated RNA. For instance, we show that the tumor-associated human repeat human satellite repeat II (HSATII) is enriched in motifs containing CpG dinucleotides in AU-rich contexts that most of the human genome and human adapted viruses have evolved to avoid. We demonstrate that a key subset of these ncRNAs functions as immunostimulatory “self-agonists” and directly activates cells of the mononuclear phagocytic system to produce proinflammatory cytokines. These ncRNAs arise from endogenous repetitive elements that are normally silenced, yet are often very highly expressed in cancers. We propose that the innate response in tumors may partially originate from direct interaction of immunogenic ncRNAs expressed in cancer cells with innate pattern recognition receptors, and thereby assign a previously unidentified danger-associated function to a set of dark matter repetitive elements. These findings potentially reconcile several observations concerning the role of ncRNA expression in cancers and their relationship to the tumor microenvironment.

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.