Caspase-8 deficiency in T cells leads to a lethal lymphoinfiltrative immune disorder

Caspase-8 is best known for its cell death function via death receptors. Recent evidence indicates that caspase-8 also has nonapoptotic functions. Caspase-8 deficiency is associated with pathologies that are unexpected for a proapoptotic molecule, such as abrogation of activation-induced lymphocyte proliferation, perturbed immune homeostasis, and immunodeficiency. In this study, we report the long-term physiological consequences of T cell–specific deletion of caspase-8 (tcasp8−/−). We show that tcasp8−/− mice develop an age-dependent lethal lymphoproliferative and lymphoinfiltrative immune disorder characterized by lymphoadenopathy, splenomegaly, and accumulation of T cell infiltrates in the lungs, liver, and kidneys. Peripheral casp8−/− T cells manifest activation marker up-regulation and are proliferating in the absence of any infection or stimulation. We also provide evidence suggesting that this immune disorder is different from the autoimmune lymphoproliferative syndrome. Interestingly, the condition described in tcasp8−/− mice manifests features consistent with the disorder described in humans with Caspase-8 deficiency. These findings suggest that tcasp8−/− mice may serve as an animal model to evaluate Caspase-8–deficient patient prognosis and therapy. Overall, our study uncovers novel in vivo functions for caspase-8 in immune regulation

Caspase-8 Inactivation in T Cells Increases Necroptosis and Suppresses Autoimmunity in Bim−/−Bim^{−/−} Mice

Dysregulation of either the extrinsic or intrinsic apoptotic pathway can lead to various diseases including immune disorders and cancer. In addition to its role in the extrinsic apoptotic pathway, caspase-8 plays nonapoptotic functions and is essential for T cell homeostasis. The pro-apoptotic BH3-only Bcl-2 family member Bim is important for the intrinsic apoptotic pathway and its inactivation leads to autoimmunity that is further exacerbated by loss of function of the death receptor Fas. We report that inactivation of caspase-8 in T cells of $Bim^{−/−}$ mice restrained their autoimmunity and extended their life span. We show that, similar to $caspase-8^{−/−}$ T cells, $Bim^{−/−}$ T cells that also lack caspase-8 displayed elevated levels of necroptosis and that inhibition of this cell death process fully rescued the survival and proliferation of these cells. Collectively, our data demonstrate that inactivation of caspase-8 suppresses the survival and proliferative capacity of $Bim^{−/−}$ T cells and restrains autoimmunity in $Bim^{−/−}$ mice

The Tumor Suppressor Gene Brca1 Is Required for Embryonic Cellular Proliferation in the Mouse

AbstractMutations of the BRCA1 gene in humans are associated with predisposition to breast and ovarian cancers. We show here that Brca1+/− mice are normal and fertile and lack tumors by age eleven months. Homozygous Brca15-6 mutant mice die before day 7.5 of embryogenesis. Mutant embryos are poorly developed, with no evidence of mesoderm formation. The extraembryonic region is abnormal, but aggregation with wild-type tetraploid embryos does not rescue the lethality. In vivo, mutant embryos do not exhibit increased apoptosis but show reduced cell proliferation accompanied by decreased expression of cyclin E and mdm-2, a regulator of p53 activity. The expression of cyclin-dependent kinase inhibitor p21 is dramatically increased in the mutant embryos. Buttressing these in vivo observations is the fact that mutant blastocyst growth is grossly impaired in vitro. Thus, the death of Brca15-6 mutant embryos prior to gastrulation may be due to a failure of the proliferative burst required for the development of the different germ layers

Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer

Signaling and repair of DNA double-strand breaks (DSBs) are critical for preventing immunodeficiency and cancer. These DNA breaks result from exogenous and endogenous DNA insults but are also programmed to occur during physiological processes such as meiosis and immunoglobulin heavy chain (IgH) class switch recombination (CSR). Recent studies reported that the E3 ligase RNF8 plays important roles in propagating DNA DSB signals and thereby facilitating the recruitment of various DNA damage response proteins, such as 53BP1 and BRCA1, to sites of damage. Using mouse models for Rnf8 mutation, we report that Rnf8 deficiency leads to impaired spermatogenesis and increased sensitivity to ionizing radiation both in vitro and in vivo. We also demonstrate the existence of alternative Rnf8-independent mechanisms that respond to irradiation and accounts for the partial recruitment of 53bp1 to sites of DNA damage in activated Rnf8−/− B cells. Remarkably, IgH CSR is impaired in a gene dose-dependent manner in Rnf8 mutant mice, revealing that these mice are immunodeficient. In addition, Rnf8−/− mice exhibit increased genomic instability and elevated risks for tumorigenesis indicating that Rnf8 is a novel tumor suppressor. These data unravel the in vivo pleiotropic effects of Rnf8

CD28-dependent Activation of Protein Kinase B/Akt Blocks Fas-mediated Apoptosis by Preventing Death-inducing Signaling Complex Assembly

The T cell costimulatory molecule CD28 is important for T cell survival, yet both the signaling pathways downstream of CD28 and the apoptotic pathways they antagonize remain poorly understood. Here we demonstrate that CD4+ T cells from CD28-deficient mice show increased susceptibility to Fas-mediated apoptosis via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. Protein kinase B (PKBα/Akt1) is an important serine/threonine kinase that promotes survival downstream of PI3K signals. To understand how PI3K-mediated signals downstream of CD28 contribute to T cell survival, we examined Fas-mediated apoptosis in T cells expressing an active form of PKBα. Our data demonstrate that T cells expressing active PKB are resistant to Fas-mediated apoptosis in vivo and in vitro. PKB transgenic T cells show reduced activation of caspase-8, BID, and caspase-3 due to impaired recruitment of procaspase-8 to the death-inducing signaling complex (DISC). Similar alterations are seen in T cells from mice which are haploinsufficient for PTEN, a lipid phosphatase that regulates phosphatidylinositol-3,4,5-trisphosphate (PIP3) and influences PKBα activity. These findings provide a novel link between CD28 and an important apoptosis pathway in vivo, and demonstrate that PI3K/PKB signaling prevents apoptosis by inhibiting DISC assembly

Th-MYCN Mice with Caspase-8 Deficiency Develop Advanced Neuroblastoma with Bone Marrow Metastasis

Neuroblastoma, the most common extracranial pediatric solid tumor, is responsible for 15% of all childhood cancer deaths. Patients frequently present at diagnosis with metastatic disease, particularly to the bone marrow (BM). Advances in therapy and understanding of the metastatic process have been limited due in part, to the lack of animal models harboring BM disease. The widely employed transgenic model, the Th-MYCN mouse, exhibits limited metastasis to this site. Here we establish the first genetic immunocompetent mouse model for metastatic neuroblastoma with enhanced secondary tumors in the BM. This model recapitulates two frequent alterations in metastatic neuroblasoma, over-expression of MYCN and loss of caspase-8 expression. Mouse caspase-8 gene was deleted in neural crest lineage cells by crossing a Th-Cre transgenic mouse with a caspase-8 conditional knockout mouse. This mouse was then crossed with the neuroblastoma prone Th-MYCN mouse. While over-expression of MYCN by itself rarely caused bone marrow metastasis, combining MYCN overexpression and caspase-8 deletion significantly enhanced BM metastasis (37% incidence). Microarray expression studies of the primary tumors mRNAs and microRNAs revealed extracellular matrix (ECM) structural changes, increased expression of genes involved in epithelial to mesenchymal transition, inflammation and down-regulation of miR-7a and miR-29b. These molecular changes have been shown to be associated with tumor progression and activation of the cytokine transforming growth factor beta (TGF-β) pathway in various tumor models. Cytokine TGF-β can preferentially promote single cell motility and blood borne metastasis and therefore activation of this pathway may explain the enhanced BM metastasis observed in this animal model.Fil: Teitz, Tal. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Inoue, Madoka. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Valentine, Marcus B.. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Zhu, Kejin. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Rehg, Jerold E.. St. Jude Children’s Research Hospital. Department of Pathology; Estados UnidosFil: Zhao, Wei. St. Jude Children’s Research Hospital. Department of Biostatistics; Estados UnidosFil: Finkelstein, David. St. Jude Children’s Research Hospital. Department of Computational Biology; Estados UnidosFil: Wang, Yong-Dong. St. Jude Children’s Research Hospital. Hartwell Center for Bioinformatics and Biotechnology; Estados UnidosFil: Johnson, Melissa D.. St. Jude Children’s Research Hospital. Animal Imaging Center; Estados UnidosFil: Calabrese, Christopher. St. Jude Children’s Research Hospital. Animal Imaging Center; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Hakem, Razqallah. University of Toronto. Ontario Cancer Institute. Department of Medical Biophysics; CanadáFil: Weiss, William A.. University of California. Departments of Neurology, Pediatrics and Neurological Surgery; Estados UnidosFil: Lahti, Jill M.. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados Unido

Inactivation of Chk2 and Mus81 Leads to Impaired Lymphocytes Development, Reduced Genomic Instability, and Suppression of Cancer

Chk2 is an effector kinase important for the activation of cell cycle checkpoints, p53, and apoptosis in response to DNA damage. Mus81 is required for the restart of stalled replication forks and for genomic integrity. Mus81Δex3-4/Δex3-4 mice have increased cancer susceptibility that is exacerbated by p53 inactivation. In this study, we demonstrate that Chk2 inactivation impairs the development of Mus81Δex3-4/Δex3-4 lymphoid cells in a cell-autonomous manner. Importantly, in contrast to its predicted tumor suppressor function, loss of Chk2 promotes mitotic catastrophe and cell death, and it results in suppressed oncogenic transformation and tumor development in Mus81Δex3-4/Δex3-4 background. Thus, our data indicate that an important role for Chk2 is maintaining lymphocyte development and that dual inactivation of Chk2 and Mus81 remarkably inhibits cancer

Excessive transcription-replication conflicts are a vulnerability of BRCA1-mutant cancers

BRCA1 mutations are associated with increased breast and ovarian cancer risk. BRCA1-mutant tumors are high-grade, recurrent, and often become resistant to standard therapies. Herein, we performed a targeted CRISPR-Cas9 screen and identified MEPCE, a methylphosphate capping enzyme, as a synthetic lethal interactor of BRCA1. Mechanistically, we demonstrate that depletion of MEPCE in a BRCA1-deficient setting led to dysregulated RNA polymerase II (RNAPII) promoter-proximal pausing, R-loop accumulation, and replication stress, contributing to transcription-replication collisions. These collisions compromise genomic integrity resulting in loss of viability of BRCA1-deficient cells. We also extend these findings to another RNAPII-regulating factor, PAF1. This study identifies a new class of synthetic lethal partners of BRCA1 that exploit the RNAPII pausing regulation and highlight the untapped potential of transcription-replication collision-inducing factors as unique potential therapeutic targets for treating cancers associated with BRCA1 mutations

Genomic Instability, Defective Spermatogenesis, Immunodeficiency, and Cancer in a Mouse Model of the RIDDLE Syndrome

Eukaryotic cells have evolved to use complex pathways for DNA damage signaling and repair to maintain genomic integrity. RNF168 is a novel E3 ligase that functions downstream of ATM,γ-H2A.X, MDC1, and RNF8. It has been shown to ubiquitylate histone H2A and to facilitate the recruitment of other DNA damage response proteins, including 53BP1, to sites of DNA break. In addition, RNF168 mutations have been causally linked to the human RIDDLE syndrome. In this study, we report that Rnf168−/− mice are immunodeficient and exhibit increased radiosensitivity. Rnf168−/− males suffer from impaired spermatogenesis in an age-dependent manner. Interestingly, in contrast to H2a.x−/−, Mdc1−/−, and Rnf8−/− cells, transient recruitment of 53bp1 to DNA double-strand breaks was abolished in Rnf168−/− cells. Remarkably, similar to 53bp1 inactivation, but different from H2a.x deficiency, inactivation of Rnf168 impairs long-range V(D)J recombination in thymocytes and results in long insertions at the class-switch junctions of B-cells. Loss of Rnf168 increases genomic instability and synergizes with p53 inactivation in promoting tumorigenesis. Our data reveal the important physiological functions of Rnf168 and support its role in both γ-H2a.x-Mdc1-Rnf8-dependent and -independent signaling pathways of DNA double-strand breaks. These results highlight a central role for RNF168 in the hierarchical network of DNA break signaling that maintains genomic integrity and suppresses cancer development in mammals