ABSTRACT
Although Ras/mitogen-activated protein kinase (MAPK) signaling is activated in most human cancers, attempts to target this pathway using kinase-active site inhibitors have not typically led to durable clinical benefit. To address this shortcoming, we sought to test the feasibility of an alternative targeting strategy, focused on the ERK2 substrate binding domains, D and DEF binding pocket (DBP). Disabling the ERK2-DBP domain in mice caused baseline erythrocytosis. Consequently, we investigated the role of the ERK2-D and -DBP domains in disease, using a JAK2-dependent model of polycythemia vera (PV). Of note, inactivation of the ERK2-DBP domain promoted the progression of disease from PV to myelofibrosis, suggesting that the ERK2-DBP domain normally opposes progression. ERK2-DBP inactivation also prevented oncogenic JAK2 kinase (JAK2V617F) from promoting oncogene-induced senescence in vitro. The ERK2-DBP mutation attenuated JAK2-mediated oncogene-induced senescence by preventing the physical interaction of ERK2 with the transcription factor Egr1. Because inactivation of the ERK2-DBP created a functional ERK2 kinase limited to binding substrates through its D domain, these data suggested that the D domain substrates were responsible for promoting oncogene-induced progenitor growth and tumor progression and that pharmacologic targeting of the ERK2-D domain may attenuate cancer cell growth. Indeed, pharmacologic agents targeting the ERK2-D domain were effective in attenuating the growth of JAK2-dependent myeloproliferative neoplasm cell lines. Taken together, these data indicate that the ERK-D and -DBP domains can play distinct roles in the progression of neoplasms and that the D domain has the potential to be a potent therapeutic target in Ras/MAPK-dependent cancers.
Subject(s)
Janus Kinase 2 , Polycythemia Vera , Animals , Cell Line , Humans , Janus Kinase 2/genetics , MAP Kinase Signaling System , Mice , Mitogen-Activated Protein Kinases , Phosphorylation , Signal TransductionABSTRACT
A novel application for non-thermal plasma is the induction of immunogenic cancer cell death for cancer immunotherapy. Cells undergoing immunogenic death emit danger signals which facilitate anti-tumor immune responses. Although pathways leading to immunogenic cell death are not fully understood; oxidative stress is considered to be part of the underlying mechanism. Here; we studied the interaction between dielectric barrier discharge plasma and cancer cells for oxidative stress-mediated immunogenic cell death. We assessed changes to the intracellular oxidative environment after plasma treatment and correlated it to emission of two danger signals: surface-exposed calreticulin and secreted adenosine triphosphate. Plasma-generated reactive oxygen and charged species were recognized as the major effectors of immunogenic cell death. Chemical attenuators of intracellular reactive oxygen species successfully abrogated oxidative stress following plasma treatment and modulated the emission of surface-exposed calreticulin. Secreted danger signals from cells undergoing immunogenic death enhanced the anti-tumor activity of macrophages. This study demonstrated that plasma triggers immunogenic cell death through oxidative stress pathways and highlights its potential development for cancer immunotherapy.
Subject(s)
Apoptosis/immunology , Immunotherapy/methods , Neoplasms/therapy , Oxidative Stress/immunology , Plasma Gases/therapeutic use , Reactive Oxygen Species/immunology , A549 Cells , Adenosine Triphosphate/metabolism , Analysis of Variance , Antigen-Presenting Cells/metabolism , Calreticulin/metabolism , Electric Conductivity , Electrodes , Humans , Immunity , Quartz/chemistry , Reactive Oxygen Species/metabolism , Ultraviolet RaysABSTRACT
Newborn screening for severe combined immunodeficiency (SCID) has not only accelerated diagnosis and improved treatment for affected infants, but also led to identification of novel genes required for human T cell development. A male proband had SCID newborn screening showing very low T cell receptor excision circles (TRECs), a biomarker for thymic output of nascent T cells. He had persistent profound T lymphopenia, but normal numbers of B and natural killer (NK) cells. Despite an allogeneic hematopoietic stem cell transplant from his brother, he failed to develop normal T cells. Targeted resequencing excluded known SCID genes; however, whole exome sequencing (WES) of the proband and parents revealed a maternally inherited X-linked missense mutation in MED14 (MED14V763A), a component of the mediator complex. Morpholino (MO)-mediated loss of MED14 function attenuated T cell development in zebrafish. Moreover, this arrest was rescued by ectopic expression of cDNA encoding the wild type human MED14 ortholog, but not by MED14V763A , suggesting that the variant impaired MED14 function. Modeling of the equivalent mutation in mouse (Med14V769A) did not disrupt T cell development at baseline. However, repopulation of peripheral T cells upon competitive bone marrow transplantation was compromised, consistent with the incomplete T cell reconstitution experienced by the proband upon transplantation with bone marrow from his healthy male sibling, who was found to have the same MED14V763A variant. Suspecting that the variable phenotypic expression between the siblings was influenced by further mutation(s), we sought to identify genetic variants present only in the affected proband. Indeed, WES revealed a mutation in the L1 cell adhesion molecule (L1CAMQ498H); however, introducing that mutation in vivo in mice did not disrupt T cell development. Consequently, immunodeficiency in the proband may depend upon additional, unidentified gene variants.
Subject(s)
Lymphopenia , Severe Combined Immunodeficiency , Animals , Humans , Infant , Infant, Newborn , Lymphopenia/genetics , Male , Mice , Neonatal Screening , Severe Combined Immunodeficiency/diagnosis , Severe Combined Immunodeficiency/genetics , Severe Combined Immunodeficiency/therapy , T-Lymphocytes , ZebrafishABSTRACT
The CD40 receptor and its ligand CD40L is one of the most critical molecular pairs of the stimulatory immune checkpoints. Both CD40 and CD40L have a membrane form and a soluble form generated by proteolytic cleavage or alternative splicing. CD40 and CD40L are widely expressed in various types of cells, among which B cells and myeloid cells constitutively express high levels of CD40, and T cells and platelets express high levels of CD40L upon activation. CD40L self-assembles into functional trimers which induce CD40 trimerization and downstream signaling. The canonical CD40/CD40L signaling is mediated by recruitment of TRAFs and NF-κB activation, which is supplemented by signal pathways such as PI3K/AKT, MAPKs and JAK3/STATs. CD40/CD40L immune checkpoint leads to activation of both innate and adaptive immune cells via two-way signaling. CD40/CD40L interaction also participates in regulating thrombosis, tissue inflammation, hematopoiesis and tumor cell fate. Because of its essential role in immune activation, CD40/CD40L interaction has been regarded as an attractive immunotherapy target. In recent years, significant advance has been made in CD40/CD40L-targeted therapy. Various types of agents, including agonistic/antagonistic monoclonal antibodies, cellular vaccines, adenoviral vectors and protein antagonist, have been developed and evaluated in early-stage clinical trials for treating malignancies, autoimmune diseases and allograft rejection. In general, these agents have demonstrated favorable safety and some of them show promising clinical efficacy. The mechanisms of benefits include immune cell activation and tumor cell lysis/apoptosis in malignancies, or immune cell inactivation in autoimmune diseases and allograft rejection. This review provides a comprehensive overview of the structure, processing, cellular expression pattern, signaling and effector function of CD40/CD40L checkpoint molecules. In addition, we summarize the progress, targeted diseases and outcomes of current ongoing and completed clinical trials of CD40/CD40L-targeted therapy.
Subject(s)
Autoimmune Diseases , Neoplasms , CD40 Antigens , CD40 Ligand , Humans , Neoplasms/drug therapy , Phosphatidylinositol 3-KinasesABSTRACT
West Nile virus (WNV) is an emerging mosquito-borne flavivirus, related to dengue virus and Zika virus. To gain insight into host pathways involved in WNV infection, we performed a systematic affinity-tag purification mass spectrometry (APMS) study to identify 259 WNV-interacting human proteins. RNA interference screening revealed 26 genes that both interact with WNV proteins and influence WNV infection. We found that WNV, dengue and Zika virus capsids interact with a conserved subset of proteins that impact infection. These include the exon-junction complex (EJC) recycling factor PYM1, which is antiviral against all three viruses. The EJC has roles in nonsense-mediated decay (NMD), and we found that both the EJC and NMD are antiviral and the EJC protein RBM8A directly binds WNV RNA. To counteract this, flavivirus infection inhibits NMD and the capsid-PYM1 interaction interferes with EJC protein function and localization. Depletion of PYM1 attenuates RBM8A binding to viral RNA, suggesting that WNV sequesters PYM1 to protect viral RNA from decay. Together, these data suggest a complex interplay between the virus and host in regulating NMD and the EJC.
Subject(s)
Antiviral Agents/pharmacology , Flavivirus Infections/drug therapy , Viral Proteins/genetics , West Nile virus/drug effects , West Nile virus/genetics , Capsid Proteins , Carrier Proteins , Codon, Nonsense , Dengue Virus/genetics , Exons , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/physiology , Humans , Protein Interaction Maps , RNA Interference , RNA, Viral , RNA-Binding Proteins , Viral Proteins/physiology , West Nile virus/pathogenicity , Zika Virus/geneticsABSTRACT
IMPORTANCE: Low-socioeconomic urban children often do not have access to ophthalmic care. OBJECTIVE: To characterize the demographic characteristics and ophthalmic conditions in children attending Give Kids Sight Day (GKSD), an outreach ophthalmic care program held annually in Philadelphia, Pennsylvania, providing vision screening and immediate treatment when needed. DESIGN, SETTING, AND PARTICIPANTS: Retrospective case-series study of children attending GKSD in 2012 (GKSD 2012) at an ophthalmology center in Philadelphia. Registration forms and records of all children attending GKSD 2012 were reviewed. MAIN OUTCOMES AND MEASURES: Demographic characteristics, insurance status, spoken languages, reasons for attending, prior failure of vision screening, and attendance pattern of previous events were analyzed. The ophthalmological findings of these children were examined, including refractive errors, need for optical correction, and diagnoses for which continuous ophthalmic care was necessary. For children who needed ophthalmic follow-up, the rate of return to clinic and barriers for continuous care were analyzed. RESULTS: We studied 924 children (mean age, 9 years; age range, 0-18 years; 51% female; 25% speaking a non-English language) coming from 584 families who attended GKSD 2012, of whom 27% were uninsured and 10% were not aware of their insurance status. Forty-two percent of participants had public insurance, which covered vision care and glasses, but 35% did not know their benefits and did not realize vision care was covered. Forty-nine percent of children attended because they failed community vision screening. Provision of free glasses and failure of previous vision screening were the most common reasons families elected to attend GKSD (64% and 49%, respectively). Eighty-five percent of children attended GKSD 2012 for the first time, whereas 15% attended prior events. Glasses were provided to 61% of attendees. Ten percent of the attendees needed continuous ophthalmic care, most commonly for amblyopia. Ten children needed ocular surgery for cataract, strabismus, nystagmus, ptosis, or nasolacrimal duct obstruction. With the assistance of a social worker, 59% of children requiring continuous treatment returned to the clinic, compared with 2% in prior years before social worker intervention. CONCLUSIONS AND RELEVANCE: Programs such as GKSD can bridge the gap between successful vision screening and ophthalmic treatment, a gap that often occurs in low-socioeconomic urban populations. Those with public insurance coverage for vision services may not realize these services are covered. Social worker intervention is useful in overcoming common barriers to follow-up care.