Endogenous IL-27 during toxoplasmosis limits early monocyte responses and their inflammatory activation by pathological T cells.

Mice that lack the genes for IL-27, or the IL-27 receptor, and infected with Toxoplasma gondii develop T cell-mediated pathology. Here, studies were performed to determine the impact of endogenous IL-27 on the immune response to T. gondii in wild-type (WT) mice. Analysis of infected mice revealed the early production of IL-27p28 by a subset of Ly6Chi, inflammatory monocytes, and sustained IL-27p28 production at sites of acute and chronic infection. Administration of anti-IL-27p28 prior to infection resulted in an early (day 5) increase in levels of macrophage and granulocyte activation, as well as enhanced effector T cell responses, as measured by both cellularity, cytokine production, and transcriptional profiling. This enhanced acute response led to immune pathology, while blockade during the chronic phase of infection resulted in enhanced T cell responses but no systemic pathology. In the absence of IL-27, the enhanced monocyte responses observed at day 10 were a secondary consequence of activated CD4+ T cells. Thus, in WT mice, IL-27 has distinct suppressive effects that impact innate and adaptive immunity during different phases of this infection. IMPORTANCE: The molecule IL-27 is critical in limiting the immune response to the parasite Toxoplasma gondii. In the absence of IL-27, a lethal, overactive immune response develops during infection. However, when exactly in the course of infection this molecule is needed was unclear. By selectively inhibiting IL-27 during this parasitic infection, we discovered that IL-27 was only needed during, but not prior to, infection. Additionally, IL-27 is only needed in the active areas in which the parasite is replicating. Finally, our work found that a previously unstudied cell type, monocytes, was regulated by IL-27, which contributes further to our understanding of the regulatory networks established by this molecule.

SRF617 Is a Potent Inhibitor of CD39 with Immunomodulatory and Antitumor Properties.

CD39 (ENTPD1) is a key enzyme responsible for degradation of extracellular ATP and is upregulated in the tumor microenvironment (TME). Extracellular ATP accumulates in the TME from tissue damage and immunogenic cell death, potentially initiating proinflammatory responses that are reduced by the enzymatic activity of CD39. Degradation of ATP by CD39 and other ectonucleotidases (e.g., CD73) results in extracellular adenosine accumulation, constituting an important mechanism for tumor immune escape, angiogenesis induction, and metastasis. Thus, inhibiting CD39 enzymatic activity can inhibit tumor growth by converting a suppressive TME to a proinflammatory environment. SRF617 is an investigational, anti-CD39, fully human IgG4 Ab that binds to human CD39 with nanomolar affinity and potently inhibits its ATPase activity. In vitro functional assays using primary human immune cells demonstrate that inhibiting CD39 enhances T-cell proliferation, dendritic cell maturation/activation, and release of IL-1ß and IL-18 from macrophages. In vivo, SRF617 has significant single-agent antitumor activity in human cell line-derived xenograft models that express CD39. Pharmacodynamic studies demonstrate that target engagement of CD39 by SRF617 in the TME inhibits ATPase activity, inducing proinflammatory mechanistic changes in tumor-infiltrating leukocytes. Syngeneic tumor studies using human CD39 knock-in mice show that SRF617 can modulate CD39 levels on immune cells in vivo and can penetrate the TME of an orthotopic tumor, leading to increased CD8+ T-cell infiltration. Targeting CD39 is an attractive approach for treating cancer, and, as such, the properties of SRF617 make it an excellent drug development candidate.

Non-Redundant Roles of T Cell Costimulation Pathways in Inflammatory Arthritis Revealed by Dual Blockade of ICOS and CD28 with Acazicolcept (ALPN-101).

OBJECTIVE: CD28 and inducible T cell costimulator (ICOS) appear to have nonredundant roles in T cell activation and adaptive immunity. We undertook this study to characterize in vitro and in vivo the therapeutic potential of acazicolcept (ALPN-101), an Fc fusion protein of a human variant ICOS ligand (ICOSL) domain designed to inhibit both CD28 and ICOS costimulation, in inflammatory arthritis. METHODS: Acazicolcept was compared in vitro with inhibitors of either the CD28 or ICOS pathways (abatacept and belatacept [CTLA-4Ig], prezalumab [anti-ICOSL monoclonal antibody]) in receptor binding and signaling assays, and in a collagen-induced arthritis (CIA) model. Acazicolcept was also compared in cytokine and gene expression assays of peripheral blood mononuclear cells (PBMCs) from healthy donors or rheumatoid arthritis (RA) or psoriatic arthritis (PsA) patients stimulated with artificial antigen-presenting cells (APCs) expressing CD28 and ICOS ligands*. RESULTS: Acazicolcept bound CD28 and ICOS, prevented ligand binding, and inhibited human T cell functional interactions, matching or exceeding the activity of CD28 or ICOS costimulatory single-pathway inhibitors tested individually or in combination. Acazicolcept administration significantly reduced disease in the CIA model and more potently than abatacept. Acazicolcept also inhibited proinflammatory cytokine production from stimulated PBMCs in cocultures with artificial APCs and demonstrated unique effects on gene expression distinct from those induced by abatacept, prezalumab, or a combination of both. CONCLUSION: Both CD28 and ICOS signaling play critical roles in inflammatory arthritis. Therapeutic agents such as acazicolcept that coinhibit both ICOS and CD28 signaling may mitigate inflammation and/or disease progression in RA and PsA more effectively than inhibitors of either pathway alone.

Povetacicept, an Enhanced Dual APRIL/BAFF Antagonist That Modulates B Lymphocytes and Pathogenic Autoantibodies for the Treatment of Lupus and Other B Cell-Related Autoimmune Diseases.

OBJECTIVE: Dysregulated APRIL/BAFF signaling is implicated in the pathogenesis of multiple autoimmune diseases, including systemic lupus erythematosus and lupus nephritis. We undertook this study to develop and evaluate a high-affinity APRIL/BAFF antagonist to overcome the clinical limitations of existing B cell inhibitors. METHODS: A variant of TACI-Fc generated by directed evolution showed enhanced binding for both APRIL and BAFF and was designated povetacicept (ALPN-303). Povetacicept was compared to wild-type (WT) TACI-Fc and related molecules in vitro and in vivo. RESULTS: Povetacicept inhibited APRIL and BAFF more effectively than all evaluated forms of WT TACI-Fc and selective APRIL and BAFF inhibitors in cell-based reporter assays and primary human B cell assays, mediating potent suppression of B cell proliferation, differentiation, and immunoglobulin (Ig) secretion. In mouse immunization models, povetacicept significantly reduced serum immunoglobulin titers and antibody-secreting cells more effectively than anti-CD20 monoclonal antibodies, WT TACI-Fc, or APRIL and BAFF inhibitors. In the NZB × NZW mouse lupus nephritis model, povetacicept significantly enhanced survival and suppressed proteinuria, anti-double-stranded DNA antibody titers, blood urea nitrogen, glomerulonephritis, and renal immunoglobulin deposition. In the bm12 mouse lupus model, povetacicept significantly reduced splenic plasmablasts, follicular helper T cells, and germinal center B cells. In non-human primates, povetacicept was well tolerated, exhibited high serum exposure, and significantly decreased serum IgM, IgA, and IgG levels after a single dose. CONCLUSION: Enhanced APRIL and BAFF inhibition by povetacicept led to greater inhibition of B cell populations critical for autoantibody production compared to WT TACI-Fc and CD20-, APRIL-, or BAFF-selective inhibitors. Potent, dual inhibition by povetacicept has the potential to significantly improve clinical outcomes in autoantibody-related autoimmune diseases.

Perceived Clinical Characteristics of Children With History of Opioid Exposure: A Speech-Language Pathology Perspective.

PURPOSE: This research was conducted to describe the clinical characteristics of children with a history of opioid exposure as perceived by the speech-language pathologists (SLPs) treating them. METHOD: Three focus groups were conducted. Participants consisted of 20 SLPs working in the schools in West Virginia who had experienced working with children with a confirmed or suspected history of opioid exposure. A thematic, qualitative analysis was conducted, whereby focus group sessions were transcribed verbatim and information was coded, organized into themes, and interpreted. RESULTS: Themes of perceived clinical characteristics (speech, language, executive function, and other developmental delays) are reported to address the research question. Additionally, themes derived from the data regarding perceived significant differentiators (greater severity/needs, inconsistent performance, and atypical manifestation) and perceived confounding characteristics (safety and well-being, aspects of home environment, and effects on school environment) that are often reported in children with a history or suspected history of opioid exposure are presented. CONCLUSIONS: Perceived clinical characteristics of this population, both intrinsic and situational, highlight the complex profile of this population and demonstrate the importance of considering each child from a multidimensional perspective. Additional research is needed to represent the profile of these children more completely and to identify successful supports that will improve their speech and language outcomes, educational achievement, and their overall quality of life.

Perceptions of Speech-Language Pathologists' Service Provision in the Opioid Epidemic: A Focus Group Study.

PURPOSE: This research intended to identify current practices being implemented with children who have a history or suspected history of opioid exposure, as well as challenges faced by speech-language pathologists in the schools. METHOD: Focus group data from three groups totaling 20 speech-language pathologists (SLPs) working in schools in West Virginia, also used in a previous study, were analyzed using qualitative thematic analysis to better understand SLP service provision to children with a history or suspected history of opioid exposure. RESULTS: Results revealed two primary themes, reported as (a) service delivery in action (current practice trends and challenges) and (b) affective/cognitive manifestations (uncertainty about their role and various emotions expressed) of the SLPs who participated in the focus groups. CONCLUSIONS: This study revealed important clinical implications derived from the reports of participants. Particularly, the importance of communication and advocacy in the care of these children and the need to rethink what our scope of practice means when working with this vulnerable population are discussed.

Potential Oncogenic Effect of the MERTK-Dependent Apoptotic-Cell Clearance Pathway in Starry-Sky B-Cell Lymphoma.

The histological architecture of certain aggressive B-cell lymphomas (prototypically Burkitt's lymphoma, BL) is characterized by a "starry-sky" (SS) appearance. This is caused by tumor-associated macrophages (TAMs), which appear in standard histological preparations as "stars" in a darkly stained "sky" of lymphoma cells. SS-TAMs accumulate in response to constitutive apoptosis in these tumors and are activated by the apoptotic tumor cells to a pro-oncogenic phenotype. The extent to which SS-TAMs contribute to lymphoma growth through responses generated by interactions with apoptotic tumor cells is unknown. Here, we demonstrate a role for the receptor tyrosine kinase, MERTK, in the oncogenic activity of SS-TAMs. We show that MERTK expression is largely restricted to the macrophages of human BL and of murine models of SS B-cell lymphoma and that it is upregulated in SS-TAMs as compared to the germinal center or paracortical macrophages of normal lymph nodes. Our results further demonstrate that MERTK is active in the phagocytosis of apoptotic lymphoma cells by macrophages and, most significantly, that SS lymphoma growth is markedly inhibited in Mertk-/- mice. These results point toward the MERTK apoptotic-cell clearance/response pathway playing a key role in growth of aggressive B-cell lymphoma and identifies MERTK as a novel potential antilymphoma target.

The Fully human anti-CD47 antibody SRF231 exerts dual-mechanism antitumor activity via engagement of the activating receptor CD32a.

BACKGROUND: CD47 is a broadly expressed cell surface glycoprotein associated with immune evasion. Interaction with the inhibitory receptor signal regulatory protein alpha (SIRPα), primarily expressed on myeloid cells, normally serves to restrict effector function (eg, phagocytosis and immune cell homeostasis). CD47/SIRPα antagonists, commonly referred to as 'macrophage checkpoint' inhibitors, are being developed as cancer interventions. SRF231 is an investigational fully human IgG4 anti-CD47 antibody that is currently under evaluation in a phase 1 clinical trial. The development and preclinical characterization of SRF231 are reported here. METHODS: SRF231 was characterized in assays designed to probe CD47/SIRPα blocking potential and effects on red blood cell (RBC) phagocytosis and agglutination. Additionally, SRF231-mediated phagocytosis and cell death were assessed in macrophage:tumor cell in vitro coculture systems. Further mechanistic studies were conducted within these coculture systems to ascertain the dependency of SRF231-mediated antitumor activity on Fc receptor engagement vs CD47/SIRPα blockade. In vivo, SRF231 was evaluated in a variety of hematologic xenograft models, and the mechanism of antitumor activity was assessed using cytokine and macrophage infiltration analyses following SRF231 treatment. RESULTS: SRF231 binds CD47 and disrupts the CD47/SIRPα interaction without causing hemagglutination or RBC phagocytosis. SRF231 exerts antitumor activity in vitro through both phagocytosis and cell death in a manner dependent on the activating Fc-gamma receptor (FcγR), CD32a. Through its Fc domain, SRF231 engagement with macrophage-derived CD32a serves dual purposes by eliciting FcγR-mediated phagocytosis of cancer cells and acting as a scaffold to drive CD47-mediated death signaling into tumor cells. Robust antitumor activity occurs across multiple hematologic xenograft models either as a single agent or in combination with rituximab. In tumor-bearing mice, SRF231 increases tumor macrophage infiltration and induction of the macrophage cytokines, mouse chemoattractant protein 1 and macrophage inflammatory protein 1 alpha. Macrophage depletion results in diminished SRF231 antitumor activity, underscoring a mechanistic role for macrophage engagement by SRF231. CONCLUSION: SRF231 elicits antitumor activity via apoptosis and phagocytosis involving macrophage engagement in a manner dependent on the FcγR, CD32a.

Apo2L/TRAIL and the death receptor 5 agonist antibody AMG 655 cooperate to promote receptor clustering and antitumor activity.

Death receptor agonist therapies have exhibited limited clinical benefit to date. Investigations into why Apo2L/TRAIL and AMG 655 preclinical data were not predictive of clinical response revealed that coadministration of Apo2L/TRAIL with AMG 655 leads to increased antitumor activity in vitro and in vivo. The combination of Apo2L/TRAIL and AMG 655 results in enhanced signaling and can sensitize Apo2L/TRAIL-resistant cells. Structure determination of the Apo2L/TRAIL-DR5-AMG 655 ternary complex illustrates how higher order clustering of DR5 is achieved when both agents are combined. Enhanced agonism generated by combining Apo2L/TRAIL and AMG 655 provides insight into the limited efficacy observed in previous clinical trials and suggests testable hypotheses to reconsider death receptor agonism as a therapeutic strategy.

Death receptor agonist therapies for cancer, which is the right TRAIL?

The activation of cell-surface death receptors represents an attractive therapeutic strategy to promote apoptosis of tumor cells. Several investigational therapeutics that target this extrinsic pathway, including recombinant human Apo2L/TRAIL and monoclonal agonist antibodies directed against death receptors-4 (DR4) or -5 (DR5), have been evaluated in the clinic. Although Phase 1/1b studies provided encouraging preliminary results, findings from randomized Phase 2 studies failed to demonstrate significant clinical benefit. This has raised multiple questions as to why pre-clinical data were not predictive of clinical response. Results from clinical studies and insight into why current agents have failed to yield robust responses are discussed. In addition, new strategies for the development of next generation death receptor agonists are reviewed.

The TRPM8 channel forms a complex with the 5-HT(1B) receptor and phospholipase D that amplifies its reversal of pain hypersensitivity.

Effective relief from chronic hypersensitive pain states remains an unmet need. Here we report the discovery that the TRPM8 ion channel, co-operating with the 5-HT(1B) receptor (5-HT(1B)R) in a subset of sensory afferents, exerts an influence at the spinal cord level to suppress central hypersensitivity in pain processing throughout the central nervous system. Using cell line models, ex vivo rat neural tissue and in vivo pain models, we assessed functional Ca(2+) fluorometric responses, protein:protein interactions, immuno-localisation and reflex pain behaviours, with pharmacological and molecular interventions. We report 5-HT(1B)R expression in many TRPM8-containing afferents and direct interaction of these proteins in a novel multi-protein signalling complex, which includes phospholipase D1 (PLD1). We provide evidence that the 5-HT(1B)R activates PLD1 to subsequently activate PIP 5-kinase and generate PIP2, an allosteric enhancer of TRPM8, achieving a several-fold increase in potency of TRPM8 activation. The enhanced activation responses of synaptoneurosomes prepared from spinal cord and cortical regions of animals with a chronic inflammatory pain state are inhibited by TRPM8 activators that were applied in vivo topically to the skin, an effect potentiated by co-administered 5-HT(1B)R agonists and attenuated by 5-HT(1B)R antagonists, while 5-HT(1B)R agents alone had no detectable effect. Corresponding results are seen when assessing reflex behaviours in inflammatory and neuropathic pain models. Control experiments with alternative receptor/TRP channel combinations reveal no such synergy. Identification of this novel receptor/effector/channel complex and its impact on nociceptive processing give new insights into possible strategies for enhanced analgesia in chronic pain.

Attenuated PLD1 association and signalling at the H452Y polymorphic form of the 5-HT(2A) receptor.

The 5-HT2A receptor (5-HT2AR) is implicated in psychotropic changes within the central nervous system (CNS). A number of polymorphisms have been reported in the 5-HT2AR gene; one of these results in a non-synonymous change, H452Y, in the carboxy-terminal tail of the receptor protein. The minor allele (9% occurrence) has been statistically associated with CNS dysfunction such as impaired memory processing and resistance to neuroleptic treatment in schizophrenic patients. We investigated the impact of H452Y mutation of the 5-HT2AR expressed in COS7 cells on distinctly coupled intracellular signalling pathways from the receptor, focusing on the heterotrimeric G protein-independent phospholipase D (PLD) pathway, compared to the conventional Gq/11-linked phospholipase C (PLC) pathway. The H452Y mutation selectively attenuated PLD signalling, which as in the wild-type receptor, was mediated by a molecular complex involving PLD1 docked to the receptor's carboxy-terminal tail domain. Co-immunoprecipitation and GST-fusion protein experiments revealed that the H452Y mutation selectively reduced PLD1 binding to the receptor. Experiments with blocking peptides to mimic short sections of the 5-HT2AR tail sequence revealed that the peptide spanning residue 452 strongly reduced PLD but not PLC responses of the receptor. Similar observations were made when assessing both PLD responses and PLD-dependent cellular proliferation elicited by activation of 5-HT2ARs natively expressed in MCF-7 cells. Overall these findings indicate that the H452Y polymorphic variant of the 5-HT2AR displays selective disruption of its PLD signalling pathway. This may potentially play a role in the CNS dysfunction associated with the H452Y allele of the 5-HT2AR.

Targeting Apo2L/TRAIL receptors by soluble Apo2L/TRAIL.

The early observation that Apo2L/TRAIL preferentially triggers apoptosis in tumor cells over normal cells highlighted its potential as a candidate therapeutic in cancer. Since its identification in the mid-1990s, our increased understanding of Apo2L/TRAIL and Apo2L/TRAIL receptor signaling has led to the development of several agonists designed to promote tumor cell apoptosis through death receptor engagement. Recombinant human Apo2L/TRAIL/dulanermin is unique in that it is the only agonist which binds both Apo2L/TRAIL death receptors. In pre-clinical studies dulanermin demonstrates broad spectrum anti-tumor activity and the ability to cooperate with multiple conventional and targeted therapies. Results from early stage clinical trials indicate that dulanermin is well tolerated and shows some evidence of clinical activity. Not all tumors are likely to be equally sensitive to apoptosis induction by Apo2L/TRAIL. Therefore, an increased understanding of the regulation of Apo2L/TRAIL signaling should aid in the identification of molecular signatures that define a patient population likely to respond. In this review, current knowledge and new insights about Apo2L/TRAIL signaling is discussed with the focus on the development of Apo2L/TRAIL as a cancer therapeutic.

5-HT2A receptor signalling through phospholipase D1 associated with its C-terminal tail.

The 5-HT2AR (5-hydroxytryptamine-2A receptor) is a GPCR (G-protein-coupled receptor) that is implicated in the actions of hallucinogens and represents a major target of atypical antipsychotic agents. In addition to its classical signalling though PLC (phospholipase C), the receptor can activate several other pathways, including ARF (ADP-ribosylation factor)-dependent activation of PLD (phospholipase D), which appears to be achieved through a mechanism independent of heterotrimeric G-proteins. In the present study we show that wild-type and inactive constructs of PLD1 (but not PLD2) respectively facilitate and inhibit ARF-dependent PLD signalling by the 5-HT2AR. Furthermore we demonstrate that PLD1 specifically co-immunoprecipitates with the receptor and binds to a distal site in GST (glutathione transferase) fusion protein constructs of its C-terminal tail which is distinct from the ARF-interaction site, thereby suggesting the existence of a functional ARF-PLD signalling complex directly associated with this receptor. This reveals the spatial co-ordination of an important GPCR, transducer and effector into a physical complex that is likely to reinforce the impact of receptor activation on a heterotrimeric G-protein-independent signalling pathway. Signalling of this receptor through such non-canonical pathways may be important to its role in particular disorders.

Death receptor agonists as a targeted therapy for cancer.

Apoptosis is integral to normal, physiologic processes that regulate cell number and results in the removal of unnecessary or damaged cells. Apoptosis is frequently dysregulated in human cancers, and recent advancements in our understanding of the regulation of programmed cell death pathways has led to the development of novel agents to reactivate apoptosis in malignant cells. The activation of cell surface death receptors by tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and death receptor agonists represent an attractive therapeutic strategy to promote apoptosis of tumor cells through the activation of the extrinsic pathway. The observation that Apo2L/TRAIL can eliminate tumor cells preferentially over normal cells has resulted in several potential therapeutics that exploit the extrinsic pathway, in particular, the soluble recombinant human (rh)Apo2L/TRAIL protein and agonist monoclonal antibodies that target death receptors 4 or 5. Many of these agents are currently being evaluated in phase 1 or 2 trials, either as a single agent or in combination with cytotoxic chemotherapy or other targeted agents. The opportunities and challenges associated with the development of death receptor agonists as cancer therapeutics, the status of ongoing clinical evaluations, and the progress toward identifying predictive biomarkers for patient selection and pharmacodynamic markers of response are reviewed.

Combined therapy with the RANKL inhibitor RANK-Fc and rhApo2L/TRAIL/dulanermin reduces bone lesions and skeletal tumor burden in a model of breast cancer skeletal metastasis.

In bone metastases, tumor cells interact with the bone microenvironment to induce osteoclastogenesis, leading to bone destruction and the growth factor release.  RANK ligand (RANKL) is essential for osteoclast formation, function, and survival.  Tumor cell-mediated osteolysis is thought to occur ultimately via induction of RANKL within the bone stroma, and inhibition of RANKL in models of breast cancer bone metastases blocks tumor-induced osteolysis and reduces skeletal tumor burden.  In addition, the skeleton is co-opted by tumor cells and functions as a supportive tumor microenvironment.  Inhibition of RANKL, by reducing tumor-induced osteoclastogenesis, may reduce the local release of growth factors and calcium which may potentially enhance the anti-tumor activity of cytotoxic or direct tumor apoptotic agents.  Recombinant human Apo2 ligand/ TNF-related apoptosis-inducing ligand (rhApo2L/TRAIL/dulanermin) is a dual pro-apoptotic receptor agonist that preferentially induces apoptosis in cancer cells versus normal cells.  We therefore examined RANKL inhibition (using RANK-Fc) in combination with rhApo2L/TRAIL on tumor-induced osteolysis and skeletal tumor burden in a murine intracardiac injection model of MDA-MB-231 breast carcinoma bone metastasis.  rhApo2L/TRAIL treatment resulted in a rapid reduction of skeletal tumor burden. Treatment with RANK-Fc prevented osteolytic lesions and reduced skeletal tumor burden. Combining RANK-Fc with rhApo2L/TRAIL was superior to either rhApo2L/TRAIL or RANK-Fc alone at reducing skeletal tumor burden in the bone metastasis model.  Our findings show that RANKL inhibition effectively inhibits pathologic osteolysis induced by human breast adenocarcinoma MDA-MB-231 cells in animals with established tumors, and also enhances the ability of rhApo2L/TRAIL to reduce skeletal tumor burden in vivo.

RIP4 regulates epidermal differentiation and cutaneous inflammation.

The receptor-interacting protein (RIP) family kinase RIP4 interacts with protein kinase C (PKC) isoforms and is implicated in PKC-dependent signaling pathways. RIP4(-/-) mice die at birth with epidermal differentiation defects, causing fusions of all external orifices and loss of the esophageal lumen. To further understand RIP4 function in the skin, we generated transgenic mice with epidermal-specific expression of RIP4 using the human keratin-14 promoter (K14-RIP4). The K14-RIP4 transgene rescued the epidermal phenotype of RIP4(-/-) mice, showing that RIP4 acts autonomously in the epidermis to regulate differentiation. Although RIP4(-/-) mice share many phenotypic similarities with inhibitor kappaB kinase (IKK)alpha(-/-) mice and stratifin repeated epilation (Sfn(Er/Er)) mice, the K14-RIP4 transgene failed to promote epidermal differentiation in these mutant backgrounds. Unexpectedly, topical treatment of K14-RIP4 mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced dramatic, neutrophilic inflammation, an effect that was independent of tumor necrosis factor type 1 receptor (TNFR1/p55) function. Despite their enhanced sensitivity to TPA, K14-RIP4 mice did not have an altered frequency of tumor formation in TPA-promoted skin cancer initiated with 7,12-dimethylbenz[a]anthracene (DMBA). These data suggest that RIP4 functions in the epidermis through PKC-specific signaling pathways to regulate differentiation and inflammation.

Ligand-based targeting of apoptosis in cancer: the potential of recombinant human apoptosis ligand 2/Tumor necrosis factor-related apoptosis-inducing ligand (rhApo2L/TRAIL).

Cancer is a leading cause of premature human death and commands considerable research attention. Apoptosis (type 1 programmed cell death) is critical in maintaining tissue homeostasis in metazoan organisms, and its dysregulation underpins the initiation and progression of cancer. Conventional chemotherapy and radiotherapy can induce apoptosis as a secondary consequence of inflicting cell damage. However, more direct and selective strategies to manipulate the apoptotic process in cancer cells are emerging as potential therapeutic tools. Genetic and biochemical understanding of the cellular signaling mechanisms that control apoptosis has increased substantially during the last decade. These advances provide a strong scientific framework for developing several types of targeted proapoptotic anticancer therapies. One promising class of agents is the proapoptotic receptor agonists. Of these, recombinant human apoptosis ligand 2/tumor necrosis factor-related apoptosis-inducing ligand (rhApo2L/TRAIL)-an optimized soluble form of an endogenous apoptosis-inducing ligand-is unique in that it activates two related proapoptotic receptors, DR4 and DR5. Preclinical data indicate that rhApo2L/TRAIL can induce apoptosis in a broad range of human cancer cell lines while sparing most normal cell types. In vitro, and in various in vivo tumor xenograft models, rhApo2L/TRAIL exhibits single-agent antitumor activity and/or cooperation with certain conventional and targeted therapies. Preclinical safety studies in nonhuman primates show rhApo2L/TRAIL to be well tolerated. Moreover, early clinical trial data suggest that rhApo2L/TRAIL is generally safe and provide preliminary evidence for potential antitumor activity. Clinical studies are ongoing to assess the safety and efficacy of this novel agent in combination with established anticancer therapies.

Expression of anti-apoptotic factors modulates Apo2L/TRAIL resistance in colon carcinoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) selectively induces apoptosis in transformed cells. Normal cells and certain tumor cells can evade Apo2L/TRAIL induced cell death, but the determinants of Apo2L/TRAIL sensitivity are poorly understood. To better understand the factors that contribute to Apo2L/TRAIL resistance, we characterized two colon carcinoma lines with pronounced differences in Apo2L/TRAIL sensitivity. Colo205 cells are highly sensitive to Apo2L/TRAIL whereas Colo320 cells are unresponsive. Components of the DISC (death inducing signaling complex) could be immunoprecipitated from both cell lines in response to Apo2L/TRAIL. Sensitizing agents including a proteasome inhibitor conferred Apo2L/TRAIL sensitivity in Colo320 cells, indicating that the apoptotic machinery was intact and functional. We specifically suppressed the expression of Bcl-2, FLIP or XIAP in Colo320 cells. Downregulation of either FLIP or XIAP but not Bcl-2 restored sensitivity of Colo320 cells to Apo2L/TRAIL. Moreover, stable knockdown of XIAP expression in Colo320 subcutaneous tumors resulted in suppression of tumor growth and sensitivity to Apo2L/TRAIL in vivo. Our results indicate that only a specific subset of anti-apoptotic proteins can confer resistance to Apo2L/TRAIL in Colo320 cells. Elucidation of the factors that contribute to Apo2L/TRAIL resistance in tumor cells may provide insight into combination therapies with Apo2L/TRAIL in a clinical setting.

Role of the conserved NPxxY motif of the 5-HT2A receptor in determining selective interaction with isoforms of ADP-ribosylation factor (ARF).

In this study we have shown that N376 to D mutation in the conserved NPxxY motif within the carboxy terminal tail domain (CT) of the 5-HT2A receptor alters the binding preference of GST-fusion protein constructs of the CT domain from ARF1 to an alternative isoform, ARF6. These findings were corroborated by experiments investigating co-immunoprecipitation of the wild type (WT) and N376D mutant of the 5-HT2A receptor with ARF1 or 6 or dominant negative ARF1/6 constructs co-expressed in COS7 cells. In functional assays of 5-HT-induced phospholipase D (PLD) activation responses of the WT receptor were inhibited by a dominant negative mutant of ARF1 but not ARF6, whereas responses of the N376D mutant were strongly inhibited by negative mutant ARF6. No equivalent effect of the ARF mutants was seen on phospholipase C activation. In experiments assaying 5-HT-induced increases in [35S]GTPgammaS binding to ARF 1/6 immunoprecipitates as a measure of ARF activation, increased ARF6 activation was seen only with the mutant receptor. When cellular PLD responses of other NPxxY- or a DPxxY-containing GPCRs were measured in the presence of dominant negative ARF1/6 constructs, the majority, but not all, fitted the pattern exemplified by the 5-HT2A receptor and its N376D mutant. These data suggest that the presence of the N or a D in this highly conserved motif is an important, but not exclusive, determinant of which ARF isoform interacts with the GPCR.