Mechanism of co-operation of mutant IL-7Rα and mutant NRAS in acute lymphoblastic leukemia: role of MYC.

Acute lymphoblastic leukemia (ALL) is an aggressive leukemia which can be derived from either T-cell or B-cell precursors. With current treatments, the survival rate is high, but the treatments are highly toxic with severe side effects. Individual mutations in IL7Rssand RAS pathways have been previously shown to be prevalent in ALL and especially in relapsed patients. The relationship of IL-7R77and RAS was investigated by transducing immature mouse thymocytes with the combination of these mutants. The resultant ALL cells were analyzed to identify the regulators and the oncoproteins that are upregulated or downregulated by the combination of IL7Rα with NRAS. Leukemia cells showed a significant increase in IL7Rw-mediated BCL2 expression, and an increase in MYC protein levels, was mainly induced by NRAS signaling. MYC was both necessary and sufficient to replace mutant NRAS and drugs targeting the MYC pathway showed a therapeutic benefit in IL-7R7/NRAS T-ALL. We suggest that MYC protein stability can be regulated by PLK-1 kinase, which was increased mainly by the NRAS signal. These studies identify novel pathways of oncogenesis and new targets for intervention that could lead to better therapeutic development.

A Focused Review of Ras Guanine Nucleotide-Releasing Protein 1 in Immune Cells and Cancer.

Four Ras guanine nucleotide-releasing proteins (RasGRP1 through 4) belong to the family of guanine nucleotide exchange factors (GEFs). RasGRPs catalyze the release of GDP from small GTPases Ras and Rap and facilitate their transition from an inactive GDP-bound to an active GTP-bound state. Thus, they regulate critical cellular responses via many downstream GTPase effectors. Similar to other RasGRPs, the catalytic module of RasGRP1 is composed of the Ras exchange motif (REM) and Cdc25 domain, and the EF hands and C1 domain contribute to its cellular localization and regulation. RasGRP1 can be activated by a diacylglycerol (DAG)-mediated membrane recruitment and protein kinase C (PKC)-mediated phosphorylation. RasGRP1 acts downstream of the T cell receptor (TCR), B cell receptors (BCR), and pre-TCR, and plays an important role in the thymocyte maturation and function of peripheral T cells, B cells, NK cells, mast cells, and neutrophils. The dysregulation of RasGRP1 is known to contribute to numerous disorders that range from autoimmune and inflammatory diseases and schizophrenia to neoplasia. Given its position at the crossroad of cell development, inflammation, and cancer, RASGRP1 has garnered interest from numerous disciplines. In this review, we outline the structure, function, and regulation of RasGRP1 and focus on the existing knowledge of the role of RasGRP1 in leukemia and other cancers.

IL-7: Comprehensive review.

OVERVIEW: IL-7 is a member of the family of cytokines with four anti-parallel α helixes that bind Type I cytokine receptors. It is produced by stromal cells and is required for development and homeostatic survival of lymphoid cells. GENOMIC ARCHITECTURE: Interleukin 7 (IL7) human IL7: gene ID: 3574 on ch 8; murine Il7 gene ID: 16,196 on ch 3. PROTEIN: Precursor contains a signal sequence, mature human IL-7 peptide 152aa, predicted 17.4kd peptide, glycosylated resulting in 25kd. Crystal structure: http://www.rcsb.org/structure/3DI2. REGULATION OF IL-7 PRODUCTION: Major producers are stromal cells in thymus, bone marrow and lymphoid organs but also reported in other tissues. Production is primarily constitutive but reported to be affected by IFNγ and other factors. IL-7 RECEPTORS: Two chains IL-7Rα (IL-7R) and γc (IL-2RG). Human IL-7R: gene ID 3575 on ch 5; human IL2RG: gene ID 3561 on ch X; mouse IL-7R: gene ID 16,197 on ch 15; murine Il2rg gene ID 16,186 on ch X. Member of γc family of receptors for cytokines IL-2, -4, -9, -15, and -21. Primarily expressed on lymphocytes but reports of other cell types. Expression in T-cells downregulated by IL-7. Low expression on Tregs, no expression on mature B-cells. Crystal structure: http://www.rcsb.org/structure/3DI2. IL-7 RECEPTOR SIGNAL TRANSDUCTION PATHWAYS: Major signals through JAK1, JAK3 to STAT5 and through non-canonical STAT3, STAT1, PI3K/AKT and MEK/ERK pathways. BIOLOGICAL ACTIVITY OF IL-7: Required for survival of immature thymocytes, naïve T-cells, memory T-cells, pro-B-cells and innate lymphocytes. Pharmacological treatment with IL-7 induces expansion of naïve and memory T-cells and pro-B-cells. ABNORMALITIES OF THE IL-7 PATHWAY IN DISEASE: Deficiencies in the IL-7 pathway in humans and mice result in severe combined immunodeficiency due to lymphopenia. Excessive signaling of the pathway in mice drives autoimmune diseases and in humans is associated with autoimmune syndromes including multiple sclerosis, type 1 diabetes, rheumatoid arthritis, sarcoidosis, atopic dermatitis and asthma. Mutations in the IL-7 receptor pathway drive acute lymphoblastic leukemia. CLINICAL APPLICATIONS: IL-7 has been evaluated in patients with cancer and shown to expand lymphocytes. It accelerated lymphocyte recovery after hematopoietic stem cell transfer, and increased lymphocyte counts in AIDS patients and sepsis patients. Monoclonal antibodies blocking the IL-7 receptor are being evaluated in autoimmune diseases. Cytotoxic monoclonals are being evaluated in acute lymphoblastic leukemia. Drugs blocking the signal transduction pathway are being tested in autoimmunity and acute lymphoblastic leukemia.

Nuphar lutea Extracts Exhibit Anti-Viral Activity against the Measles Virus.

Different parts of Nuphar lutea L. (yellow water lily) have been used to treat several inflammatory and pathogen-related diseases. It has shown that Nuphar lutea extracts (NUP) are active against various pathogens including bacteria, fungi, and leishmanial parasites. In an effort to detect novel therapeutic agents against negative-stranded RNA (- RNA) viruses, we have tested the effect of a partially-purified alkaloid mixture of Nuphar lutea leaves on the measles virus (MV). The MV vaccine's Edmonston strain was used to acutely or persistently infect cells. The levels of several MV proteins were detected by a Western blot and immunocytochemistry. Viral RNAs were quantitated by qRT-PCR. Virus infectivity was monitored by infecting African green monkey kidney VERO cells' monolayers. We showed that NUP protected cells from acute infection. Decreases in the MV P-, N-, and V-proteins were observed in persistently infected cells and the amount of infective virus released was reduced as compared to untreated cells. By examining viral RNAs, we suggest that NUP acts at the post-transcriptional level. We conclude, as a proof of concept, that NUP has anti-viral therapeutic activity against the MV. Future studies will determine the mechanism of action and the effect of NUP on other related viruses.

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia.

Transduced mouse immature thymocytes can be differentiated into T cells in vitro using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

Mutations that collaborate with IL-7Ra signaling pathways to drive ALL.

The IL-7 pathway is required for normal T cell development and survival. In recent years the pathway has been shown to be a major driver of acute lymphoblastic leukemia (ALL), the most common cancer in children. Gain-of-function mutations in the alpha chain of the IL-7 receptor found in ALL patients clearly demonstrated that this pathway was a driver. However mutant IL-7R alone was insufficient to transform primary T cell progenitors, indicating that cooperating mutations were required. Here we review evidence for additional oncogenic mutations in the IL-7 pathway. We discuss several oncogenes, loss of tumor suppressor genes and epigenetic effects that can cooperate with mutant IL-7 receptor. These include NRas, HOXA, TLX3, Notch 1, Arf, PHF6, WT1, PRC, PTPN2 and CK2. As new therapeutics targeting the IL-7 pathway are developed, combination with agents directed to cooperating pathways offer hope for novel therapies for ALL.

Autophagy differentially regulates TNF receptor Fn14 by distinct mammalian Atg8 proteins.

Autophagy, a conserved membrane trafficking process, sequesters cytoplasmic components into autophagosomes and targets them for lysosomal degradation. The TNF receptor Fn14 participates in multiple intracellular signaling pathways and is strongly induced upon tissue injury and solid tumorigenesis. While Fn14 is a short-lived protein, the regulation of its levels is largely obscure. Here we uncover a role for autophagy in Fn14 turnover, wherein specific core autophagy Atg8 proteins play distinct roles: Fn14 accumulates in the ERGIC in absence of GABARAP but within endosomes in the vicinity of autophagic membranes in absence of GATE-16. Moreover, GABARAP regulates overall cellular levels of Fn14, whereas GATE-16 regulates TWEAK signaling by Fn14 and thereby NF-κB activity. These findings not only implicate different Atg8 proteins in distinct roles within the mechanism of selective autophagic regulation of Fn14, but may also provide a more general view of their role in mediating autophagosome biogenesis from different membrane sources.