Jean M. Veith

Curaxins: Anticancer Compounds That Simultaneously Suppress NF-κB and Activate p53 by Targeting FACT

The quinacrine-related compounds curaxins target multiple procancer pathways through FACT complex. Curaxins: Cancer Therapy Grounded in FACT Targeted cancer therapies offer the possibility of personalized therapies with reduced toxicity, but their impact is limited by the development of drug resistance and subsequent proliferation of tumor cells that are refractory to further treatment. Combination therapies might help overcome the resistance problem because a tumor cell is less likely to be simultaneously resistant to multiple drugs that act by distinct mechanisms, but the potential for negative drug interactions and increased toxicities causes concern in the clinic. Here, Gasparian et al. kill two birds with one stone: They find that the quinacrine-related DNA-intercalating compounds curaxins can target multiple procancer pathways with minimal toxicity. Curaxins are small molecules that simultaneously activate p53 and inhibit nuclear factor κB (NF-κB), two pathways that are altered in diverse tumor types. These drugs show strong anticancer activity in mice without detectable genotoxicity. Here, Gasparian et al. determine the mechanism behind curaxins’ success. These molecules trap the FACT (facilitates chromatin transcription) complex within chromatin, which results in p53 phosphorylation and inhibition of NF-κB–dependent transcription. This study not only supports a role for curaxins as potentially safe agents that target multiple pathways involved in diverse cancer types but also promotes FACT as a prime target for future bimodal therapies. Although it remains to be seen whether these data are reproducible in humans, defining curaxins’ mechanism of action is a major step in translating these promising small molecules to the clinic. Effective eradication of cancer requires treatment directed against multiple targets. The p53 and nuclear factor κB (NF-κB) pathways are dysregulated in nearly all tumors, making them attractive targets for therapeutic activation and inhibition, respectively. We have isolated and structurally optimized small molecules, curaxins, that simultaneously activate p53 and inhibit NF-κB without causing detectable genotoxicity. Curaxins demonstrated anticancer activity against all tested human tumor xenografts grown in mice. We report here that the effects of curaxins on p53 and NF-κB, as well as their toxicity to cancer cells, result from “chromatin trapping” of the FACT (facilitates chromatin transcription) complex. This FACT inaccessibility leads to phosphorylation of the p53 Ser392 by casein kinase 2 and inhibition of NF-κB–dependent transcription, which requires FACT activity at the elongation stage. These results identify FACT as a prospective anticancer target enabling simultaneous modulation of several pathways frequently dysregulated in cancer without induction of DNA damage. Curaxins have the potential to be developed into effective and safe anticancer drugs.

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.

Synthesis and biological activity of novel 3′-trifluoromethyl taxoids

Second generation taxoids possessing a trifluoromethyl moiety in place of the 3′-phenyl group are synthesized by means of the β-Lactam Synthon Method. The in vitro cytotoxicities of these new taxoids are evaluated against several different human cancer cell lines and found to exhibit greatly enhanced activities as compared to those of paclitaxel and docetaxel. The activity enhancement is most remarkable against a drug-resistant breast cancer cell line, MCF7-R, expressing MDR phenotype.

SYNTHESIS AND BIOLOGICAL ACTIVITY OF 3'-ALKYL- AND 3'-ALKENYL-3'-DEPHENYLDOCETAXELS

Abstract 3-Alkyl- and 3′-alkenyl-3′-dephenyldocetaxels are synthesized from 10-deacetylbaccatin III based on the β-lactam synthon method in good yields. The cytotoxicity of the new taxoids are evaluated against different human tumor cell lines and their ability to inhibit the microtubules disassembly examined. The 3′-isobutenyl, 3′-crotyl, and 3′-isobutyl analogs possess very strong cytotoxicity as well as antitumor activity in vivo . 3′-Isobutenyl- as well as 3′-crotyl-3′-dephenyl-10-acetyldocetaxel shows ca. 20 times stronger activity against an adriamycin-resistant human breast cancer cell line (MCF7-R) than those o docetaxel and paclitaxel.

Use of an Anti-Vascular Endothelial Growth Factor Antibody in a Pharmacokinetic Strategy to Increase the Efficacy of Intraperitoneal Chemotherapy

The efficacy of intraperitoneal chemotherapy for ovarian cancers is limited by poor penetration of drug into peritoneal tumors. Based on pharmacokinetic theory that suggests that penetration depth is primarily determined by the rate of drug removal via tumor capillaries, we have hypothesized that co-administration of antiangiogenic therapy will allow for decreased drug removal, increased drug concentrations in tumor, and increased efficacy of intraperitoneal chemotherapy. Pharmacokinetic modeling was conducted to simulate the effect of tumor blood flow on tumor concentrations of topotecan. Simulations predicted that tumor blood flow reductions, as potentially achieved by antiangiogenic therapy, would lead to substantial increases in tumor concentrations after intraperitoneal chemotherapy but would lead to a slight decrease after systemic chemotherapy. Pharmacokinetic studies performed using the A2780 xenograft tumor model showed that animals receiving combined intraperitoneal topotecan and an anti-vascular endothelial growth factor (VEGF) monoclonal antibody had ∼6.5-fold higher (p = 0.0015) tumor topotecan concentrations compared with animals receiving intraperitoneal topotecan alone, whereas there was no significant (p = 0.16) difference for systemic topotecan. Therapeutic studies conducted with two different drugs, topotecan and cisplatin, showed that animals receiving combined intraperitoneal chemotherapy and anti-VEGF therapy displayed superior survival relative to animals treated with chemotherapy alone (i.e., cisplatin or topotecan), anti-VEGF alone, or intravenous chemotherapy with concomitant anti-VEGF therapy. Combined intraperitoneal topotecan and anti-VEGF resulted in the complete cure of four of 11 mice. The proposed combination of antiangiogenic therapy and intraperitoneal chemotherapy, which was predicted to be beneficial by pharmacokinetic simulations, may provide substantial benefit to patients with peritoneal malignancies.

Toll-like receptor-5 agonist, entolimod, suppresses metastasis and induces immunity by stimulating an NK-dendritic-CD8+ T-cell axis

Significance Innate immune modulators can generate a potent antitumor T-cell response and are thus a desirable approach to immunotherapy. However, their use is limited by the risk of induction of acute inflammation. In this regard, bacterial flagellin is unique among other innate immune modulators because of a significantly safer cytokine profile induced upon activation by its target, Toll-like receptor 5 (TLR5). We show here that systemic administration of entolimod, a pharmacologically optimized flagellin derivative, induces a cascade of cell–cell signaling resulting in mobilization to the liver of various components of innate and adaptive immunity, followed by suppression of liver metastases and development of long-term antitumor immune memory. Thus, TLR5 agonists can be considered as an organ-specific immunotherapy for the treatment and prevention of metastases. Activation of an anticancer innate immune response is highly desirable because of its inherent ability to generate an adaptive antitumor T-cell response. However, insufficient safety of innate immune modulators limits clinical use to topical applications. Toll-like receptor 5 (TLR5) agonists are favorably positioned as potential systemic immunotherapeutic agents because of unusual tissue specificity of expression, uniquely safe profile of induced cytokines, and antitumor efficacy demonstrated in a number of animal models. Here, we decipher the molecular and cellular events underlying the metastasis suppressive activity of entolimod, a clinical stage TLR5 agonist that activates NF-κB–, AP-1–, and STAT3–driven immunomodulatory signaling pathways specifically within the liver. Used as a single agent in murine colon and mammary metastatic cancer models, entolimod rapidly induces CXCL9 and -10 that support homing of blood-borne CXCR3-expressing NK cells to the liver predominantly through an IFN-γ signaling independent mechanism. NK cell-dependent activation of dendritic cells is followed by stimulation of a CD8+ T-cell response, which exert both antimetastatic effect of entolimod and establishment of tumor-specific and durable immune memory. These results define systemically administered TLR5 agonists as organ-specific immunoadjuvants, enabling efficient antitumor vaccination that does not depend on identification of tumor-specific antigens.

Design and synthesis of de novo cytotoxic alkaloids by mimicking the bioactive conformation of paclitaxel.

Novel paclitaxel-mimicking alkaloids were designed and synthesized based on a bioactive conformation of paclitaxel, that is, REDOR-Taxol. The alkaloid 2 bearing a 5-7-6 tricyclic scaffold mimics REDOR-Taxol best among the compounds designed and was found to be the most potent compound against several drug-sensitive and drug-resistant human cancer cell lines. MD simulation study on the paclitaxel mimics 1 and 2 as well as REDOR-Taxol bound to the 1JFF tubulin structure was quite informative to evaluate the level of mimicking. The MD simulation study clearly distinguishes the 5-6-6 and 5-7-6 tricyclic scaffolds, and also shows substantial difference in the conformational stability of the tubulin-bound structures between 2 and REDOR-Taxol. The latter may account for the large difference in potency, and provides critical information for possible improvement in the future design of paclitaxel mimics.

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.