Modifying akt signaling in B-cell chronic lymphocytic leukemia cells.

Emerging evidence suggests that the survival of B-cell chronic lymphocytic leukemia (CLL) cells is dependent on microenvironmental influences such as antigenic stimulation and support by stromal cells. Akt, also known as protein kinase B, is a central component in prosurvival signaling downstream of these events. We investigated the role of Akt and its modulation by the protooncogene T-cell leukemia 1a (Tcl1a) in the survival pathways of primary CLL samples and CLL-derived prolymphocytic cell lines MEC-1 and MEC-2. Akt activation was increased by the protective presence of human bone marrow stromal cells and B-cell receptor mimicking signals but antagonized by direct Akt blockade with the novel specific inhibitor AiX, with preferential apoptosis induction in CLL cells with an unmutated immunoglobulin status, which predicts poor clinical outcome. In addition, we found a direct interaction of Akt with Tcl1a in an endogenous coimmunoprecipitation assay. Confirming the critical role of Tcl1a in modulating Akt signaling, Akt activation was enhanced by overexpressing Tcl1a in CLL. In contrast, decreasing Tcl1a levels by small interfering RNA reduced Akt activation in the fludarabine-insensitive CLL cell line MEC-2 and sensitized the malignant cells to fludarabine treatment. In summary, our data reveal a significant role for the Akt-Tcl1a axis in CLL survival and propose a further evaluation of this interplay for targeting chemoresistance phenomena.

Interdependent regulation of p53 and miR-34a in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has an incidence 4/1,00,000 people in the western world and is one of the first cancers reported to be associated with deregulated miRNA expression. microRNAs are small non coding RNAs that are important regulators of protein expression through binding to their untranslated 3'-UTR region. The miR-34 family was demonstrated to be induced by the tumor suppressor p53 and to elicit p53-like responses like senescence, cell cycle arrest and apoptosis depending on the cell type. We have shown in a recent paper that miR-34a is severely increased in the TCL1-mouse model of CLL. This finding was reflected in human CLL. Moreover, it is demonstrated that its expression is dependent on the presence of the SNP309 in the intronic promoter of the MDM2 gene. In addition, low miR-34a expression was associated with shorter time to treatment (log-rank p = 0.003) in CLL. When reintroduced into CLL cells, miR-34a was able to induce apoptosis. Interestingly, this was dependent on an intact p53 pathway. Here, we present data showing that knockdown of p53 in HCT-116 cells severely reduces miR-34a induced apoptosis. In conclusion, miR-34a is proposed as a marker for the activity of the p53 pathway in CLL.

microRNA-34a expression correlates with MDM2 SNP309 polymorphism and treatment-free survival in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (B-CLL), aberrations along the p53 axis lead to decreased overall survival and therapy resistance. Recent studies identified microRNA-34a (miR-34a) as a major downstream target of p53. We monitored the expression of miR-34a during disease development in a murine B-CLL model. miR-34a was up-regulated more than 20-fold during the leukemic but not during the preleukemic phase. In the human system, B-CLL cells also had 4.6-fold higher miR-34a expression compared with B cells of healthy controls. In B-CLL cells of patients with p53 aberrations, miR-34a expression was consistently low. The broad distribution of miR-34a levels in p53 wild-type patients prompted us to study the correlation between single nucleotide polymorphism 309 (SNP309) in the intronic promoter of MDM2 and miR-34a expression. B-CLL cells of patients with the SNP309 GG genotype had significantly lower miR-34a expression levels compared with patients with the TT genotype (P = .002). Low miR-34a levels were able to predict shorter time to treatment (P = .003) and were associated with an abbreviated lymphocyte doubling time. Further, overexpression of miR-34a in primary B-CLL cells induced apoptosis. These findings suggest miR-34a as a possible therapeutic avenue and a sensitive indicator of the activity of the p53 axis in B-CLL.

PKCbeta is essential for the development of chronic lymphocytic leukemia in the TCL1 transgenic mouse model: validation of PKCbeta as a therapeutic target in chronic lymphocytic leukemia.

The development and the propagation of chronic lymphocytic leukemia (CLL) has been linked to signaling via the B-cell receptor (BCR). Protein kinase C beta (PKCbeta) is an essential signaling element of the BCR and was recently shown to be overexpressed in human CLL. We used the TCL1 transgenic mouse model to directly target PKCbeta in the development of murine CLL. TCL1 overexpression did restore the CD5(+) B-cell population that is absent in PKCbeta-deficient mice. However, PKCbeta-deleted TCL1 transgenic mice did not develop a CLL disease, suggesting a role of PKCbeta in the establishment of the malignant clone. Moreover, targeting of PKCbeta with the specific inhibitor enzastaurin led to killing of human CLL samples in vitro. We thus propose that PKCbeta may be a relevant target for the treatment of CLL.

Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system.

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.

Human T-cell leukemia virus type 1 envelope glycoprotein gp46 interacts with cell surface heparan sulfate proteoglycans.

The major receptors required for attachment and entry of the human T-cell leukemia virus type 1 (HTLV-1) remain to be identified. Here we demonstrate that a functional, soluble form of the HTLV-1 surface envelope glycoprotein, gp46, fused to an immunoglobulin Fc region (gp46-Fc) binds to heparan sulfate proteoglycans (HSPGs) on mammalian cells. Substantial binding of gp46-Fc to HeLa and Chinese hamster ovary (CHO) K1 cells that express HSPGs was detected, whereas binding to the sister CHO lines 2244, which expresses no HSPGs, and 2241, which expresses no glycosaminoglycans (GAGs), was much reduced. Enzymatic removal of HSPGs from HeLa and CHO K1 cells also reduced gp46-Fc binding. Dextran sulfate inhibited gp46-Fc binding to HSPG-expressing cells in a dose-dependent manner, whereas chondroitin sulfate was less effective. By contrast, dextran sulfate inhibited gp46-Fc binding to GAG-negative cells such as CHO 2244, CHO 2241, and Jurkat T cells weakly or not at all. Dextran sulfate inhibited HTLV-1 envelope glycoprotein (Env)-pseudotyped virus infection of permissive, HSPG-expressing target cells and blocked syncytium formation between HTLV-1 Env-expressing cells and HSPG-expressing permissive target cells. Finally, HSPG-expressing cells were more permissive for HTLV-1 Env-pseudotyped virus infection than HSPG-negative cells. Thus, similar to other pathogenic viruses, HTLV-1 may have evolved to use HSPGs as cellular attachment receptors to facilitate its propagation.

An antiviral peptide targets a coiled-coil domain of the human T-cell leukemia virus envelope glycoprotein.

Retrovirus entry into cells is mediated by the viral envelope glycoproteins which, through a cascade of conformational changes, orchestrate fusion of the viral and cellular membranes. In the absence of membrane fusion, viral entry into the host cell cannot occur. For human T-cell leukemia virus type 1 (HTLV-1), synthetic peptides that mimic a carboxy-terminal region of the transmembrane glycoprotein (TM) ectodomain are potent inhibitors of membrane fusion and virus entry. Here, we demonstrate that this class of inhibitor targets a fusion-active structure of HTLV-1 envelope. In particular, the peptides bind specifically to a core coiled-coil domain of envelope, and peptide variants that fail to bind the coiled-coil lack inhibitory activity. Our data indicate that the inhibitory peptides likely function by disrupting the formation of a trimer-of-hairpins structure that is required for membrane fusion. Importantly, we also show that peptides exhibiting dramatically increased potency can be readily obtained. We suggest that peptides or peptide mimetics targeting the fusion-active structures of envelope may be of therapeutic value in the treatment of HTLV-1 infections.