Erythroferrone contributes to hepcidin suppression and iron overload in a mouse model of ß-thalassemia.

Inherited anemias with ineffective erythropoiesis, such as ß-thalassemia, manifest inappropriately low hepcidin production and consequent excessive absorption of dietary iron, leading to iron overload. Erythroferrone (ERFE) is an erythroid regulator of hepcidin synthesis and iron homeostasis. Erfe expression was highly increased in the marrow and spleen of Hbb(Th3/+) mice (Th3/+), a mouse model of thalassemia intermedia. Ablation of Erfe in Th3/+ mice restored normal levels of circulating hepcidin at 6 weeks of age, suggesting ERFE could be a factor suppressing hepcidin production in ß-thalassemia. We examined the expression of Erfe and the consequences of its ablation in thalassemic mice from 3 to 12 weeks of age. The loss of ERFE in thalassemic mice led to full restoration of hepcidin mRNA expression at 3 and 6 weeks of age, and significant reduction in liver and spleen iron content at 6 and 12 weeks of age. Ablation of Erfe slightly ameliorated ineffective erythropoiesis, as indicated by reduced spleen index, red cell distribution width, and mean corpuscular volume, but did not improve the anemia. Thus, ERFE mediates hepcidin suppression and contributes to iron overload in a mouse model of ß-thalassemia.

Identification of a candidate therapeutic antibody for treatment of canine B-cell lymphoma.

B-cell lymphoma is one of the most frequently observed non-cutaneous neoplasms in dogs. For both human and canine BCL, the standard of care treatment typically involves a combination chemotherapy, e.g. "CHOP" therapy. Treatment for human lymphoma greatly benefited from the addition of anti-CD20 targeted biological therapeutics to these chemotherapy protocols; this type of therapeutic has not been available to the veterinary oncologist. Here, we describe the generation and characterization of a rituximab-like anti-CD20 antibody intended as a candidate treatment for canine B-cell lymphoma. A panel of anti-canine CD20 monoclonal antibodies was generated using a mouse hybridoma approach. Mouse monoclonal antibody 1E4 was selected for construction of a canine chimeric molecule based on its rank ordering in a flow cytometry-based affinity assay. 1E4 binds to approximately the same location in the extracellular domain of CD20 as rituximab, and 1E4-based chimeric antibodies co-stain canine B cells in flow cytometric analysis of canine leukocytes using an anti-canine CD21 antibody. We show that two of the four reported canine IgG subclasses (cIgGB and cIgGC) can bind to canine CD16a, a receptor involved in antibody-dependent cellular cytotoxicity (ADCC). Chimeric monoclonal antibodies were assembled using canine heavy chain constant regions that incorporated the appropriate effector function along with the mouse monoclonal 1E4 anti-canine CD20 variable regions, and expressed in CHO cells. We observed that 1E4-cIgGB and 1E4-cIgGC significantly deplete B-cell levels in healthy beagle dogs. The in vivo half-life of 1E4-cIgGB in a healthy dog was ∼14 days. The antibody 1E4-cIgGB has been selected for further testing and development as an agent for the treatment of canine B-cell lymphoma.

IL-2 immunotherapy reveals potential for innate beta cell regeneration in the non-obese diabetic mouse model of autoimmune diabetes.

Type-1 diabetes (T1D) is an autoimmune disease targeting insulin-producing beta cells, resulting in dependence on exogenous insulin. To date, significant efforts have been invested to develop immune-modulatory therapies for T1D treatment. Previously, IL-2 immunotherapy was demonstrated to prevent and reverse T1D at onset in the non-obese diabetic (NOD) mouse model, revealing potential as a therapy in early disease stage in humans. In the NOD model, IL-2 deficiency contributes to a loss of regulatory T cell function. This deficiency can be augmented with IL-2 or antibody bound to IL-2 (Ab/IL-2) therapy, resulting in regulatory T cell expansion and potentiation. However, an understanding of the mechanism by which reconstituted regulatory T cell function allows for reversal of diabetes after onset is not clearly understood. Here, we describe that Ab/IL-2 immunotherapy treatment, given at the time of diabetes onset in NOD mice, not only correlated with reversal of diabetes and expansion of Treg cells, but also demonstrated the ability to significantly increase beta cell proliferation. Proliferation appeared specific to Ab/IL-2 immunotherapy, as anti-CD3 therapy did not have a similar effect. Furthermore, to assess the effect of Ab/IL-2 immunotherapy well after the development of diabetes, we tested the effect of delaying treatment for 4 weeks after diabetes onset, when beta cells were virtually absent. At this late stage after diabetes onset, Ab/IL-2 treatment was not sufficient to reverse hyperglycemia. However, it did promote survival in the absence of exogenous insulin. Proliferation of beta cells could not account for this improvement as few beta cells remained. Rather, abnormal insulin and glucagon dual-expressing cells were the only insulin-expressing cells observed in islets from mice with established disease. Thus, these data suggest that in diabetic NOD mice, beta cells have an innate capacity for regeneration both early and late in disease, which is revealed through IL-2 immunotherapy.

Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids.

For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269-277]. However, the decreased ability of the immune system to mount a robust immune response to self-antigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO(2)Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-alpha (mTNF-alpha) independently to pNO(2)Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-alpha IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-alpha and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO(2)Phe(43) mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.

Immunochemical termination of self-tolerance.

The ability to selectively induce a strong immune response against self-proteins, or increase the immunogenicity of specific epitopes in foreign antigens, would have a significant impact on the production of vaccines for cancer, protein-misfolding diseases, and infectious diseases. Here, we show that site-specific incorporation of an immunogenic unnatural amino acid into a protein of interest produces high-titer antibodies that cross-react with WT protein. Specifically, mutation of a single tyrosine residue (Tyr(86)) of murine tumor necrosis factor-alpha (mTNF-alpha) to p-nitrophenylalanine (pNO(2)Phe) induced a high-titer antibody response in mice, whereas no significant antibody response was observed for a Tyr(86) --> Phe mutant. The antibodies generated against the pNO(2)Phe are highly cross-reactive with native mTNF-alpha and protect mice against lipopolysaccharide (LPS)-induced death. This approach may provide a general method for inducing an antibody response to specific epitopes of self- and foreign antigens that lead to a neutralizing immune response.

A TRAIL receptor-dependent synthetic lethal relationship between MYC activation and GSK3beta/FBW7 loss of function.

The MYC protooncogene is frequently deregulated in human cancers. Here, by screening a kinase-directed library of small inhibitory RNAs, we identify glycogen synthase kinase 3beta (GSK3beta) as a gene whose inactivation potentiates TNF-related apoptosis-inducing ligand death receptor-mediated apoptosis specifically in MYC-overexpressing cells. Small inhibitory RNA-induced silencing of GSK3beta prevents phosphorylation of MYC on T58, thereby inhibiting recognition of MYC by the E3 ubiquitin ligase component FBW7. Attenuating the GSK3beta-FBW7 axis results in stabilization of MYC, up-regulation of surface levels of the TNF-related apoptosis-inducing ligand death receptor 5, and potentiation of death receptor 5-induced apoptosis in vitro and in vivo. These results identify GSK3beta and FBW7 as potential cancer therapeutic targets and MYC as a critical substrate in the GSK3beta survival-signaling pathway. The results also demonstrate paradoxically that MYC-expressing tumors might be treatable by drug combinations that increase rather than decrease MYC oncoprotein function.

Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal recognition particle.

TRAIL (TNF-related apoptosis-inducing ligand) death receptors DR4 and DR5 facilitate the selective elimination of malignant cells through the induction of apoptosis. From previous studies the regulation of the DR4 and DR5 cell-death pathways appeared similar; nevertheless in this study we screened a library of small interfering RNA (siRNA) for genes, which when silenced, differentially affect DR4- vs. DR5-mediated apoptosis. These experiments revealed that expression of the signal recognition particle (SRP) complex is essential for apoptosis mediated by DR4, but not DR5. Selective diminution of SRP subunits by RNA interference resulted in a dramatic decrease in cell surface DR4 receptors that correlated with inhibition of DR4-dependent cell death. Conversely, SRP silencing had little influence on cell surface DR5 levels or DR5-mediated apoptosis. Although loss of SRP function in bacteria, yeast and protozoan parasites causes lethality or severe growth defects, we observed no overt phenotypes in the human cancer cells studied--even in stable cell lines with diminished expression of SRP components. The lack of severe phenotype after SRP depletion allowed us to delineate, for the first time, a mechanism for the differential regulation of the TRAIL death receptors DR4 and DR5--implicating the SRP complex as an essential component of the DR4 cell-death pathway.

Synthetic lethal targeting of MYC by activation of the DR5 death receptor pathway.

The genetic concept of synthetic lethality provides a framework for identifying genotype-selective anticancer agents. In this approach, changes in cellular physiology that arise as a consequence of oncogene activation or tumor suppressor gene loss, rather than oncoproteins themselves, are targeted to achieve tumor selectivity. Here we show that agonists of the TRAIL death receptor DR5 potently induce apoptosis in human cells overexpressing the MYC oncogene, both in vitro and as tumor xenografts in vivo. MYC sensitizes cells to DR5 in a p53-independent manner by upregulating DR5 cell surface levels and stimulating autocatalytic processing of procaspase-8. These results identify a novel mechanism by which MYC sensitizes cells to apoptosis and validate DR5 agonists as potential MYC-selective cancer therapeutics.

Activation and suppression of the TRAIL death-receptor pathway in chemotherapy sensitive and resistant follicular lymphoma cells.

Aberrant expression of the apoptosis inhibitor bcl-2 provides a survival advantage throughout oncogenesis and can facilitate chemotherapeutic resistance in a variety of human cancers. Follicular lymphoma (FL) for example, is characterized by the chromosomal translocation t(14;18), which results in bcl-2 overexpression and initiates lymphomagenesis. Although FL cells possess ample amounts of bcl-2, they respond remarkably well to standard first-round chemotherapy. However, the vast majority of patients relapses and becomes progressively resistant to therapy. We obtained cell lines derived from chemosensitive and chemoresistant FL patients, that are characterized by the chromosomal translocation t(14;18) and expression of bcl-2, to investigate how chemotherapeutic drugs can circumvent bcl-2 anti-apoptotic function and to identify alterations in those pathways that may facilitate resistance to DNA damaging drugs. In chemosensitive FL cells, we found that DNA damaging drugs promote apoptosis through p53-dependent upregulation of the TRAIL-DR5 receptor, resulting in activation of caspase-8 and downstream executioner caspases, thereby evading bcl-2 mediated suppression of apoptosis. Examination of drug resistant FL cell lines revealed that at least two defects in this pathway can contribute to chemotherapeutic resistance: 1. p53 gene mutations that disable the transcriptional response to DNA damaging drugs, including expression of the TRAIL-DR5 receptor, and 2. transcriptional repression of the cell-death executioner enzyme caspase-3.