Categorizing the characteristics of human carcinogens: a need for specificity.

The International Agency for Research on Cancer (IARC) has recently proposed employing "ten key characteristics of human carcinogens" (TKCs) to determine the potential of agents for harmful effects. The TKCs seem likely to confuse the unsatisfactory correlation from testing regimes that have ignored the differences evident when cellular changes are compared in short and long-lived species, with their very different stem cell and somatic cell phylogenies. The proposed characteristics are so broad that their use will lead to an increase in the current unacceptably high rate of false positives. It could be an informative experiment to take well-established approved therapeutics with well-known human safety profiles and test them against this new TKC paradigm. Cancers are initiated and driven by heritable and transient changes in gene expression, expand clonally, and progress via additional associated acquired mutations and epigenetic alterations that provide cells with an evolutionary advantage. The genotoxicity testing protocols currently employed and required by regulation, emphasize testing for the mutational potential of the test agent. Two-year, chronic rodent cancer bioassays are intended to test for the entire spectrum of carcinogenic transformation. The use of cytotoxic doses causing increased, sustained cell proliferation that facilitates accumulated genetic damage leads to a high false-positive rate of tumor induction. Current cancer hazard assessment protocols and weight-of-the-evidence analysis of agent-specific cancer risk align poorly with the pathogenesis of human carcinoma and so need modernization and improvement in ways suggested here.

Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis.

We have shown that cellular inhibitor of apoptosis proteins (cIAPs) impair clearance of hepatitis B virus (HBV) infection by preventing TNF-mediated killing/death of infected cells. A key question, with profound therapeutic implications, is whether this finding can be translated to the development of drugs that promote elimination of infected cells. Drug inhibitors of cIAPs were developed as cancer therapeutics to promote TNF-mediated tumor killing. These drugs are also known as Smac mimetics, because they mimic the action of the endogenous protein Smac/Diablo that antagonizes cIAP function. Here, we show using an immunocompetent mouse model of chronic HBV infection that birinapant and other Smac mimetics are able to rapidly reduce serum HBV DNA and serum HBV surface antigen, and they promote the elimination of hepatocytes containing HBV core antigen. The efficacy of Smac mimetics in treating HBV infection is dependent on their chemistry, host CD4(+) T cells, and TNF. Birinapant enhances the ability of entecavir, an antiviral nucleoside analog, to reduce viral DNA production in HBV-infected animals. These results indicate that birinapant and other Smac mimetics may have efficacy in treating HBV infection and perhaps, other intracellular infections.

Cellular inhibitor of apoptosis proteins prevent clearance of hepatitis B virus.

Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection.

Reproducibility in science: improving the standard for basic and preclinical research.

Medical and scientific advances are predicated on new knowledge that is robust and reliable and that serves as a solid foundation on which further advances can be built. In biomedical research, we are in the midst of a revolution with the generation of new data and scientific publications at a previously unprecedented rate. However, unfortunately, there is compelling evidence that the majority of these discoveries will not stand the test of time. To a large extent, this reproducibility crisis in basic and preclinical research may be as a result of failure to adhere to good scientific practice and the desperation to publish or perish. This is a multifaceted, multistakeholder problem. No single party is solely responsible, and no single solution will suffice. Here we review the reproducibility problems in basic and preclinical biomedical research, highlight some of the complexities, and discuss potential solutions that may help improve research quality and reproducibility.

Tribute to Donald Metcalf.

A collection of tributes and remembrances from esteemed colleagues, mentees, and friends on the life and work of "the father of hematopoietic cytokines".

A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates.

Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.

Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia.

Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure, and recurrence in acute myeloid leukemia (AML). Despite AML displaying extensive biological and clinical heterogeneity, LSC with high interleukin-3 receptor (IL3R) levels are a constant yet puzzling feature, as this receptor lacks tyrosine kinase activity. Here, we show that the heterodimeric IL3Rα/ßc receptor assembles into hexamers and dodecamers through a unique interface in the 3D structure, where high IL3Rα/ßc ratios bias hexamer formation. Importantly, receptor stoichiometry is clinically relevant as it varies across the individual cells in the AML hierarchy, in which high IL3Rα/ßc ratios in LSCs drive hexamer-mediated stemness programs and poor patient survival, while low ratios mediate differentiation. Our study establishes a new paradigm in which alternative cytokine receptor stoichiometries differentially regulate cell fate, a signaling mechanism that may be generalizable to other transformed cellular hierarchies and of potential therapeutic significance. SIGNIFICANCE: Stemness is a hallmark of many cancers and is largely responsible for disease emergence, progression, and relapse. Our finding that clinically significant stemness programs in AML are directly regulated by different stoichiometries of cytokine receptors represents a hitherto unexplained mechanism underlying cell-fate decisions in cancer stem cell hierarchies. This article is highlighted in the In This Issue feature, p. 1749.

Functional erythropoietin receptor is undetectable in endothelial, cardiac, neuronal, and renal cells.

Erythropoiesis stimulating agents (ESAs) have been reported to activate erythropoietin receptors (EpoR) on cell types, including endothelial, neuronal, renal tubule, and cardiac cells. ESAs have also been reported to promote angiogenesis. However, those findings are controversial and confounded by methodologic issues. We show that EpoR mRNA was detected in essentially all cell types examined, including primary human endothelial, renal, cardiac, and neuronal cells but 10- to 100-fold lower than Epo-responsive cells using quantitative reverse-transcribed polymerase chain reaction. Total endothelial EpoR protein examined using a new monoclonal antibody was low to undetectable. Surface EpoR on endothelial cells was not detected using [(125)I]-rHuEpo surface-binding studies. There was no evidence of ESA-induced intracellular signaling in endothelial cells. There was a similar lack of EpoR expression and signaling in other cell types examined. Experiments were performed examining ESA function on these cells. An in vivo rat corneal angiogenesis assay demonstrated neo-vessel formation in response to recombinant human vascular endothelial growth factor (rHuVEGF). However, recombinant mouse Epo did not induce vessel formation. Similarly, ESAs did not reproducibly provide cytoprotection to neuronal, renal, or cardiac cells. Taken together, our data challenge the notion of presence or function of EpoR on nonhematopoietic cells, and call into question the preclinical basis for clinical studies exploring direct, "pleiotropic" actions of ESAs.

Absence of functional EpoR expression in human tumor cell lines.

Certain oncology trials showed worse clinical outcomes in the erythropoiesis-stimulating agent (ESA) arm. A potential explanation was that ESA-activated erythropoietin (Epo) receptors (EpoRs) promoted tumor cell growth. Although there were supportive data from preclinical studies, those findings often used invalidated reagents and methodologies and were in conflict with other studies. Here, we further investigate the expression and function of EpoR in tumor cell lines. EpoR mRNA levels in 209 human cell lines representing 16 tumor types were low compared with ESA-responsive positive controls. EpoR protein production was evaluated in a subset of 66 cell lines using a novel anti-EpoR antibody. EpoR(+) control cells had an estimated 10 000 to 100 000 EpoR dimers/cell. In contrast, 54 of 61 lines had EpoR protein levels lower than 100 dimers/cell. Cell lines with the highest EpoR protein levels (400-3200 dimers/cell) were studied further, and, although one line, NCI-H661, bound detectable levels of [(125)I]-recombinant human Epo (rHuEpo), none showed evidence of ESA-induced EpoR activation. There was no increased phosphorylation of STAT5, AKT, ERK, or S6RP with rHuEpo. In addition, EpoR knockdown with siRNAs did not affect viability in 2 cell lines previously reported to express functional EpoR (A2780 and SK-OV-3). These results conflict with the hypothesis that EpoR is functionally expressed in tumors.

Antihepcidin antibody treatment modulates iron metabolism and is effective in a mouse model of inflammation-induced anemia.

Iron maldistribution has been implicated in multiple diseases, including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino acid peptide. Hepcidin is induced by inflammation, causes iron to be sequestered, and thus, potentially contributes to AI. Human hepcidin (hHepc) overexpression in mice caused an iron-deficient phenotype, including stunted growth, hair loss, and iron-deficient erythropoiesis. It also caused resistance to supraphysiologic levels of erythropoiesis-stimulating agent, supporting the hypothesis that hepcidin may influence response to treatment in AI. To explore the role of hepcidin in inflammatory anemia, a mouse AI model was developed with heat-killed Brucella abortus treatment. Suppression of hepcidin mRNA was a successful anemia treatment in this model. High-affinity antibodies specific for hHepc were generated, and hHepc knock-in mice were produced to enable antibody testing. Antibody treatment neutralized hHepc in vitro and in vivo and facilitated anemia treatment in hHepc knock-in mice with AI. These data indicate that antihepcidin antibodies may be an effective treatment for patients with inflammatory anemia. The ability to manipulate iron metabolism in vivo may also allow investigation of the role of iron in a number of other pathologic conditions.

Lack of expression and function of erythropoietin receptors in the kidney.

BACKGROUND: Erythropoiesis-stimulating agents (ESAs) stimulate formation of red blood cells by binding to and activating Epo receptors (EpoR) on erythroid progenitor cells. Beyond successful treatment of anemia, ESAs have been reported to reduce damage following insult to organs, including the kidney, possibly via direct activation of EpoR. However, data on ESA effects outside hematopoietic functions are conflicting. Furthermore, limited use of appropriate EpoR-positive and EpoR-negative controls and lack of specific anti-EpoR antibodies make interpretation of data difficult. Recently positive and negative control cell types were validated and a sensitive and specific anti-EpoR antibody (A82) that detects low levels of EpoR protein was described. METHODS: A82 was used to measure EpoR protein levels in tissues, human renal cells and human cell lines by western blot analysis. Surface EpoR was examined on renal cells by measuring binding of [125I]-rHuEpo or antibodies. Renal cells and cell lines were treated with rHuEpo to see if phosphorylation of signaling proteins or proliferation/survival could be induced. Small inhibitory RNA (siRNA) were used to determine if EpoR knockdown affected cell viability. RESULTS: Total EpoR protein was low to undetectable in tissues and renal cells with no detectable EpoR on cell surfaces. EpoR knockdown had no effect on viability of renal cell lines. RHuEpo had no detectable effect on intracellular signaling on renal cell lines with no growth-promoting or survival effect on primary human renal cells. CONCLUSIONS: These results suggest that functional EpoR protein is absent on renal cells and that non-EpoR mechanisms should be explored to explain non-hematopoietic effects of ESAs.

Drug repurposing: Misconceptions, challenges, and opportunities for academic researchers.

Drug repurposing is promoted as a cost- and time-effective mechanism for providing new medicines. Often, however, there is insufficient consideration by academic researchers of the processes required to ensure that a repurposed drug can be used for a new indication. This may explain the inability of drug repurposing to fulfill its promise. Important aspects, often overlooked, include financial and intellectual property considerations, the clinical and regulatory path, and clinical equipoise, which provides ethical justification for randomized controlled trials. The goal of drug repurposing is to obtain a new regulator-approved label for an existing drug, and so, the trajectory for drug repurposing and traditional drug development is similar. Here, we discuss factors critical for a successful repurposed medicine to help academic investigators better identify drug repurposing opportunities.

Identifying and understanding factors that affect the translation of therapies from the laboratory to patients: a study protocol.

Background: The process of translating preclinical findings into a clinical setting takes decades. Previous studies have suggested that only 5-10% of the most promising preclinical studies are successfully translated into viable clinical applications. The underlying determinants of this low success rate (e.g. poor experimental design, suboptimal animal models, poor reporting) have not been examined in an empirical manner. Our study aims to determine the contemporary success rate of preclinical-to-clinical translation, and subsequently determine if an association between preclinical study design and translational success/failure exists. Methods: Established systematic review methodology will be used with regards to the literature search, article screening and study selection process. Preclinical, basic science studies published in high impact basic science journals between 1995 and 2015 will be included. Included studies will focus on publicly available interventions with potential clinical promise. The primary outcome will be successful clinical translation of promising therapies - defined as the conduct of at least one Phase II trial (or greater) with a positive finding. A case-control study will then be performed to evaluate the association between elements of preclinical study design and reporting and the likelihood of successful translation. Discussion: This study will provide a comprehensive analysis of the therapeutic translation from the laboratory bench to the bedside. Importantly, any association between factors of study design and the success of translation will be identified. These findings may inform future research teams attempting preclinical-to-clinical translation. Results will be disseminated to identified knowledge users that fund/support preclinical research.

The Antibody Society's antibody validation webinar series.

In the wake of the reproducibility crisis and numerous discussions on how commercially available antibodies as research tool contribute to it, The Antibody Society developed a series of 10 webinars to address the issues involved. The webinars were delivered by speakers with both academic and commercial backgrounds. This report highlights the problems, and offers solutions to help the scientific community appropriately identify the right antibodies and to validate them for their research and development projects. Despite the various solutions proposed here, they must be applied on a case-by-case basis. Each antibody must be verified based on the content of the product sheet, and subsequently through experimentation to confirm integrity, specificity and selectivity. Verification needs to focus on the precise application and tissue/cell type for which the antibody will be used, and all verification data must be reported openly. The various approaches discussed here all have caveats, so a combination of solutions must be considered.

Functional but abnormal adult erythropoiesis in the absence of the stem cell leukemia gene.

Previous studies have indicated that the stem cell leukemia gene (SCL) is essential for both embryonic and adult erythropoiesis. We have examined erythropoiesis in conditional SCL knockout mice for at least 6 months after loss of SCL function and report that SCL was important but not essential for the generation of mature red blood cells. Although SCL-deleted mice were mildly anemic with increased splenic erythropoiesis, they responded appropriately to endogenous erythropoietin and hemolytic stress, a measure of late erythroid progenitors. However, SCL was more important for the proliferation of early erythroid progenitors because the predominant defects in SCL-deleted erythropoiesis were loss of in vitro growth of the burst-forming erythroid unit and an in vivo growth defect revealed by transplant assays. With respect to erythroid maturation, SCL-deleted proerythroblasts could generate more mature erythroblasts and circulating red blood cells. However, SCL was required for normal expression of TER119, one of the few proposed target genes of SCL. The unexpected finding that SCL-independent erythropoiesis can proceed in the adult suggests that alternate factors can replace the essential functions of SCL and raises the possibility that similar mechanisms also explain the relatively minor defects previously observed in SCL-null hematopoietic stem cells.