An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation

MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.

Application of human lymphoid cells for the evaluation of antivirals against human adenovirus type 19: Zalcitabine has superior activity compared to cidofovir

Human adenovirus type 19 (HAdV-19) is a major cause of the epidemic keratoconjunctivitis. Outbreaks of keratoconjunctivitis are problematic to human health, especially for infants, the elderly, and immunocompromised individuals. However, the development of anti-HAdV drugs has been hampered by inconvenient screening systems; therefore, development of a simple screening method is highly desirable. In this study, we identified that HAdV-19 can infect a human lymphoid cell line transformed with human T-cell leukemia virus (MT-2 cells). MT-2 cells supported HAdV-19 replication and showed apparent cytopathic effects within five days post-infection. Using a thiazolyl blue tetrazolium bromide (MTT)-based colorimetric assay on MT-2 cells, we were able to detect the anti-HAdV-19 activities of previously reported nucleoside/tide compounds, including (S)-1–(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (cidofovir), 2′,3′-dideoxycytidine (zalcitabine) and 3′-deoxy-3′-fluorothymidine (trifluridine). Compared with previous methods, this system represents a more simple and rapid method to screen anti-HAdV-19 agents.

Isoproterenol-induced beta-2 adrenergic receptor activation negatively regulates interleukin-2 signaling

Regulation of intracellular signaling pathways in lymphocytes is critical for cell homeostasis and immune response. Interleukin-2 (IL-2), a key regulator of lymphocytes, signals following receptor-ligand engagement and subsequent recruitment and activation of effector proteins including JAKs and STATs. Lymphocytes can also be regulated by the central nervous system through the β2 adrenergic receptor (β2AR) pathway which can affect cell trafficking, proliferation, differentiation, and cytokine production. The cross-talk between these two signaling pathways represents an important mechanism that has yet to be fully elucidated. The present study provides evidence for communication between the IL-2 receptor (IL-2R) and β2AR. Treatment of human lymphoid cell lines with the β2AR agonist isoproterenol (ISO) alone increased cAMP levels and mediated a stimulatory response by activating AKT and ERK to promote cell viability. Interestingly, ISO activation of β2AR also induced threonine phosphorylation of the IL-2Rβ. In contrast, ISO treatment prior to IL-2 stimulation produced an inhibitory signal that disrupted IL-2 induced activation of the JAK/STAT, MEK/ERK, and PI3K pathways by inhibiting the formation of the IL-2R beta–gamma chain complex, and subsequently cell proliferation. Moreover, γc-family cytokines-mediated STAT5 activation was also inhibited by ISO. These results suggest a molecular mechanism by which β2AR signaling can both stimulate and suppress lymphocyte responses and thus explain how certain therapeutic agents, such as vasodilators, may impact immune responsiveness.

Development of a Human Anti-CD27 Antibody with Efficacy in Lymphoma and Leukemia Models by Two Distinct Mechanisms

Abstract 2861 CD27, a lymphoid cell-specific TNFR superfamily member, is constitutively expressed on the majority of T cells, some NK cells and memory B cells. Through interaction with its ligand CD70, CD27 transduces a co-stimulatory signal promoting T cell and NK cell activation and cytotoxicity. In addition, CD27 is also expressed on many lymphoid-originated hematological neoplastic cells, such as chronic lymphocytic leukemia, mantle cell lymphoma, and Waldenstrom macroglobulinemia, thus being a potential direct target for antibody therapy. To generate potential antibodies for clinical development, we immunized human Ig transgenic mice and developed a panel of CD27 specific human mAbs. Clone 1F5 was identified as a lead based on its high affinity to both human and monkey CD27, enhanced co-stimulation of T cells, and ADCC of CD27-expressing lymphoblastic cell lines. Using SCID mice challenged with CD27-expressing human lymphoid cell lines, we demonstrated that 1F5 mediates conventional antibody effector function. Compared to human IgG1 isotype control (huIgG1), 1F5 at doses ranging 33 μg – 500 μg (x 6) significantly delayed the growth of Burkitt's lymphoma Raji even when administration was initiated 1 week after tumor inoculation. Similar anti-tumor activity was observed against other CD27-expressing tumor lines including, Daudi and T-originated acute lymphoblastic leukemia CCRF-CEM. In order to investigate 1F5 in vivo agonistic activities and T cell-mediated tumor eradication, a human CD27 transgenic mouse model (hCD27-Tg) was generated and backcrossed onto C57BL/6 and BALB/c backgrounds. The expression profile and regulation of the human CD27 transgene driven by its own promoter were similar to that observed with endogenous mouse CD27. In addition to enhancing T cell responses when combined with vaccination, 1F5 treatment was highly effective against syngeneic mouse tumors including lymphoma BCL1 (BALB/c) and thymoma EL4-derived E.G7 (C57BL/6). For the BCL1 model, various dose levels of 1F5 mAb were delivered to mice intraperitoneally on days 3, 5, 7, 9 and 11 after i.v. administration of 107 BCL1 cells to huCD27 Tg and control animals. Controls including hCD27-Tg mice treated with saline or isotype control, or WT mice treated with 1F5 all performed consistently, leading to 50% survival approximately 23 days after tumor challenge. Treatment of mice with mAb, 1F5 substantially improved the 50% survival in a dose dependent fashion to >70 days post tumor challenge at the higher dose levels. Based on the promising efficacy data with anti-CD27 mAb 1F5 in immunocompromised and immunocompetent lymphoma models, a clinical grade product, referred to as CDX-1127 was manufactured and tested for safety. To assess the potential for 1F5 to mediate lymphocyte activation, we investigated its ability to induce proliferation and cytokine release from human PBMC or purified T cell cultures. Consistent with the known biology of CD27 we demonstrated the 1F5 mAb does not lead to direct activation of lymphocytes in the absence of additional signals. However, combining 1F5 with suboptimal levels of T cell receptor stimulation using anti-CD3 mAb (OKT3) was shown to enhance proliferation of human T cells. Two studies were performed using cynomolgus macaques. There were no CDX-1127 related mortalities or changes noted in the clinical condition, food appetence, body weights and body temperature, ophthalmic, electrocardiographic and clinical pathology assessments, organ weights and bone marrow assessments. In addition, there were no major differences in the percentage of lymphocyte populations between control and CDX-1127 treated animals at the end of the study demonstrating that the antibody did not significantly deplete normal CD27-expresssing cells. Based on the pre-clinical studies we are planning a Phase 1 clinical trial of CDX-1127 in patients with hematological malignancies and selected solid tumors. The trial is designed with separate arms to independently assess the safety and activity of CDX-1127 in hematologic malignancies, in which the antibody may act through multiple mechanisms, and in solid tumors where it would be fully dependent on indirect immune mechanisms. Disclosures: He: Celldex Therapeutics, Inc.: Employment. Thomas:Celldex Therapeutics, Inc.: Employment. Weidlick:Celldex Therapeutics, Inc.: Employment. Vitale:Celldex Therapeutics, Inc.: Employment. O'Neill:Celldex Therapeutics, Inc.: Employment. Prostak:Celldex Therapeutics, Inc.: Employment. Sundarapandiyan:Celldex Therapeutics, Inc.: Employment. Marsh:Celldex Therapeutics, Inc.: Employment. Yellin:Celldex Therapeutics, Inc.: Employment. Davis:Celldex Therapeutics, Inc.: Employment. Keler:Celldex Therapeutics, Inc.: Employment.

The Cellular and Molecular Mechanisms of Neutropenia in Barth Syndrome

Abstract 1105 Barth syndrome (BTHS) is a rare, X-linked recessive disease characterized by progressive cardiomyopathy and neutropenia with life-threatening bacterial infections. BTHS is attributable to loss-of-function mutations in the tafazzin gene, TAZ, a phospholipid acyltransferase localized to the mitochondrial membrane and involved in remodeling cardiolipin. BTHS children exhibit reduced blood levels of total cardiolipin; however, the molecular and cellular mechanisms of BTHS remain largely unknown. We developed a model of BTHS by transfecting human HL60 myeloid progenitor cells with TAZ-specific shRNAs. Results demonstrated a significant down-regulation in the TAZ gene expression, mimicking naturally-occurring truncation mutations. FACS analyses of cells with TAZ-specific but not scrambled shRNAs demonstrated nearly two-fold increase in proportion of annexin-V positive cells and significantly increased dissipation of mitochondrial membrane potential (MMP) as determined by DIOC6-staining. Similar pro-apoptotic effects were observed in U937 myeloid progenitors, but not in Jurkat or Ramos human lymphoid cell lines. Further studies revealed aberrant release of cytochrome c from mitochondria and significantly elevated levels of activated caspase-3 in response to TAZ knock-down. Treatment with caspase-specific inhibitor zVAD-fmk reduced apoptosis to near-normal levels. FACS analysis utilizing cryopreserved bone marrow cells from a BTHS patient showed evidence of accelerated apoptosis compared to corresponding cells from a healthy volunteer. Importantly, human myeloid progenitor cells and cardiomyoblasts/cardiomyocytes depend heavily on mitochondria, which may explain why the loss of mitochondrial protein tafazzin has more severe effects on these specific tissues in BTHS resulting in neutropenia and cardiomyopathy. These data suggest that neutropenia in BTHS patients is attributable to increased dissipation of MMP, aberrant release of cytochrome c, activation of caspase-3 and accelerated apoptosis, and that this defect can be partially restored in vitro by treatment with caspase-specific inhibitors. Further studies should evaluate the TAZ–regulated mitochondrial apoptosis pathway using primary cells from BTHS patients. Disclosures: Aprikyan: Barth Syndrome Foundation: Research Funding. Dale:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Cytotoxicity of farnesyltransferase inhibitors in lymphoid cells mediated by MAPK pathway inhibition and Bim up-regulation

The mechanism of cytotoxicity of farnesyltransferase inhibitors is incompletely understood and seems to vary depending on the cell type. To identify potential determinants of sensitivity or resistance for study in the accompanying clinical trial (Witzig et al, page 4882), we examined the mechanism of cytotoxicity of tipifarnib in human lymphoid cell lines. Based on initial experiments showing that Jurkat variants lacking Fas-associated death domain or procaspase-8 undergo tipifarnib-induced apoptosis, whereas cells lacking caspase-9 or overexpressing Bcl-2 do not, we examined changes in Bcl-2 family members. Tipifarnib caused dose-dependent up-regulation of Bim in lymphoid cell lines (Jurkat, Molt3, H9, DoHH2, and RL) that undergo tipifarnib-induced apoptosis but not in lines (SKW6.4 and Hs445) that resist tipifarnib-induced apoptosis. Further analysis demonstrated that increased Bim levels reflect inhibition of signaling from c-Raf to MEK1/2 and ERK1/2. Additional experiments showed that down-regulation of the Ras guanine nucleotide exchange factor RasGRP1 diminished tipifarnib sensitivity, suggesting that H-Ras or N-Ras is a critical farnesylation target upstream of c-Raf in lymphoid cells. These results not only trace a pathway through c-Raf to Bim that contributes to tipifarnib cytotoxicity in human lymphoid cells but also identify potential determinants of sensitivity to this agent.