Is the c-Cbl proto-oncogene involved in chronic lymphocytic leukemia?

Chronic lymphocytic leukemia (CLL) is characterized by survival advantage and accumulation of CD5+ mature B lymphocytes. Expression of zeta-chain-associated protein-70 (ZAP-70), normally present in T lymphocytes or immature B cells, is associated with disease aggressiveness, as IgVH mutational status, and some proteins implicated in survival signal pathways are found to be constitutively activated in CLL cells. ZAP-70 signaling is regulated through molecular adaptors, such as the proto-oncogene product c-Casitas B lineage lymphoma (c-Cbl). The aim of this study was to determine the implication of this proto-oncogene product in CLL in survival signals. It appeared that expression of c-Cbl was increased in CLL and not correlated to that of B cell linker protein or ZAP-70. Furthermore, c-Cbl was significantly hypophosphorylated in progressive disease, so that hypophosphorylated form of c-Cbl (c-Cbl.P) along with ZAP-70, set a cutoff ratio distributing patients with stable situation below 1, and those with progressive disease equal or above 1. Given that phospholipase gamma 2 (PLC gamma 2) function is also influenced by c-Cbl hypophosphorylation, the ratio of PLC gamma 2 to c-Cbl.P was measured in CLL B cells and consistently found to be >or= 1 in Binet stage B CLL patients, as opposed to stage A CLL patients. These findings invite analysis of the role of c-Cbl in CLL.

Cytokine-regulated expression and inhibitory function of FcgammaRIIB1 and -B2 receptors in human dendritic cells.

Dendritic cells (DC) capture immune complexes (IC) via Fc receptors for immunoglobulin G FcgammaRII and elicit antigen presentation and protective antitumoral immune response in mice. Two protocols are commonly used to differentiate human monocyte-derived DC in vitro. They associate granulocyte macrophage-colony stimulating factor (CM-CSF) with interleukin (IL)-4 or IL-13. In this study, we first assessed the ability of the two types of DC to initiate an immune response against an IC-linked antigen. We evidenced that IL-4 and IL-13 DC display comparable lymphocyte stimulatory capacity and similar lifetimes. We next characterized FcgammaRIIs expressed by pure populations of circulating myeloid DC (BDCA1+DC), IL-4, and IL-13 DC. We highlighted the expression of FcgammaRIIA, -B1, and -B2 by pure populations of BDCA1 myeloid DCs and IL-4 and IL-13 DC. Moreover, IL-4 and IL-13 DC displayed greater FcgammaRIIB expression than monocytes but a comparable FcgammaRIIA. We next investigated the FcgammaRIIB mechanism of action. We evidenced that deleting FcgammaRIIB increased the ability of IC-pulsed DC to stimulate autologous lymphocytes. FcgammaRIIB acted by lowering IC uptake, surface expression of costimulation molecules, and cytokine release. Finally, the balance between activating FcgammaRIIA/inhibitory FcgammaRIIB (B1+B2) could be modulated in vitro by inflammation mediators. By lowering FcgammaRIIB expression without significantly affecting FcgammaRIIA, prostaglandin E2 (PGE-2) appeared to be a major regulator of this balance. IL-1beta and tumor necrosis factor alpha were also found to potentiate PGE-2 action. Altogether, our results evidence an inhibitory role for FcgammaRIIB in human DC and provide an easy way to possibly improve in vitro the induction of immune response against IC-linked antigen.

Inhibition of NF-κB-mediated signaling by the cyclin-dependent kinase inhibitor CR8 overcomes prosurvival stimuli to induce apoptosis in chronic lymphocytic leukemia cells.

PURPOSE: Chronic lymphocytic leukemia (CLL) is currently incurable with standard chemotherapeutic agents, highlighting the need for novel therapies. Overcoming proliferative and cytoprotective signals generated within the microenvironment of lymphoid organs is essential for limiting CLL progression and ultimately developing a cure. EXPERIMENTAL DESIGN: We assessed the potency of cyclin-dependent kinase (CDK) inhibitor CR8, a roscovitine analog, to induce apoptosis in primary CLL from distinct prognostic subsets using flow cytometry-based assays. CLL cells were cultured in in vitro prosurvival and proproliferative conditions to mimic microenvironmental signals in the lymphoid organs, to elucidate the mechanism of action of CR8 in quiescent and proliferating CLL cells using flow cytometry, Western blotting, and quantitative real-time PCR. RESULTS: CR8 was 100-fold more potent at inducing apoptosis in primary CLL cells than roscovitine, both in isolated culture and stromal-coculture conditions. Importantly, CR8 induced apoptosis in CD40-ligated CLL cells and preferentially targeted actively proliferating cells within these cultures. CR8 treatment induced downregulation of the antiapoptotic proteins Mcl-1 and XIAP, through inhibition of RNA polymerase II, and inhibition of NF-κB signaling at the transcriptional level and through inhibition of the inhibitor of IκB kinase (IKK) complex, resulting in stabilization of IκBα expression. CONCLUSIONS: CR8 is a potent CDK inhibitor that subverts pivotal prosurvival and proproliferative signals present in the tumor microenvironment of CLL patient lymphoid organs. Our data support the clinical development of selective CDK inhibitors as novel therapies for CLL.

Therapeutic activity of two xanthones in a xenograft murine model of human chronic lymphocytic leukemia.

BACKGROUND: We previously reported that allanxanthone C and macluraxanthone, two xanthones purified from Guttiferae trees, display in vitro antiproliferative and proapoptotic activities in leukemic cells from chronic lymphocytic leukemia (CLL) and leukemia B cell lines. RESULTS: Here, we investigated the in vivo therapeutic effects of the two xanthones in a xenograft murine model of human CLL, developed by engrafting CD5-transfected chronic leukemia B cells into SCID mice. Treatment of the animals with five daily injections of either allanxanthone C or macluraxanthone resulted in a significant prolongation of their survival as compared to control animals injected with the solvent alone (p = 0.0006 and p = 0.0141, respectively). The same treatment of mice which were not xenografted induced no mortality. CONCLUSION: These data show for the first time the in vivo antileukemic activities of two plant-derived xanthones, and confirm their potential interest for CLL therapy.