ZAP-70 is a key signaling molecule in T cells. It couples the antigen-activated T-cell receptor to downstream signaling pathways. Its expression in leukemic B-cells derived from a subgroup of patients with chronic lymphocytic leukemia (CLL) is associated with an aggressive course of the disease. However, its implication for the pathogenesis of aggressive CLL is still unclear. In this study, we show that the expression of ZAP-70 enhances the signals associated with the B-cell receptor, recruiting protein kinase C-bII (PKC-bII) into lipid raft domains. Subsequently, PKC-bII is activated and shuttles from the plasma membrane to the mitochondria. We unravel that the antiapoptotic protein Bcl-2 and its antagonistic BH3-protein Bim EL are putative substrates for PKC-bII. PKC-bII-mediated phosphorylation of Bcl-2 augments its antiapoptotic function by increasing its ability to sequester more pro-apoptotic Bim EL. In addition, the phosphorylation of Bim EL by PKC-bII leads to its proteasomal degradation. These changes confer leukemic cells to a more antiapoptotic state with aggressiveness of the disease. Most importantly, these molecular changes can be therapeutically targeted with the small molecule inhibitor Enzastaurin. We provide evidence that this compound is highly active in leukemic cells and augments the cytotoxic effects of standard chemotherapeutic drugs.

Immunotherapy with rituximab alone or in conjunction with chemotherapy has significantly improved the treatment outcome of B-cell lymphoma patients. Nevertheless, a subpopulation of patients does not respond to rituximab. The reason for treatment failure as well as the exact mechanism of action is still uncertain. The function of rituximab has long been associated with the partitioning of CD20 molecules to lipid microdomains. We now show that the extent of CD20 recruitment to lipid rafts correlates with response to rituximab. In addition, expression of the raft-associated sphingolipid GM1 on lymphoma cells is associated with the susceptibility of lymphoma cells to rituximab. Furthermore, we show substantially different GM1 expression in various primary non-Hodgkin's lymphomas. Whereas chronic lymphocytic leukemia (CLL) cells have a low GM1 expression, marginal zone lymphoma cells exhibit much higher levels. Differences were not only detected among various lymphoma subgroups but also within one lymphoma subtype. Interestingly, whereas CLL cells from patients with high GM1 expression responded to rituximab, patients with low GM1 expressing CLL cells did not. These data show the importance of membrane microdomains in the effect of rituximab and may offer a predictive factor for the responsiveness of lymphoma cells to rituximab. [Cancer Res 2008;68(13):5414-22]

Immunotherapy with rituximab alone or in conjunction with chemotherapy has significantly improved the treatment outcome of B-cell lymphoma patients. Nevertheless, a subpopulation of patients does not respond to rituximab. The reason for treatment failure as well as the exact mechanism of action is still uncertain. The function of rituximab has long been associated with the partitioning of CD20 molecules to membrane microdomains. Here, we show that concomitant antifungal treatment with itraconazole impairs the rituximab antilymphoma effect both in vitro and in vivo. At the molecular level, recruitment of CD20 to lipid rafts is inhibited in the presence of itraconazole. Furthermore, calcium influx, which is crucial for rituximab-mediated cell death, was nearly completely abolished by itraconazole treatment. In contrast, the antifungal drug caspofungin did not inhibit CD20 recruitment to lipid rafts, nor did it affect calcium influx or the cytotoxic effect of rituximab. The finding that itraconazole also abolished the cytotoxic effects of other therapeutic antibodies directed against lipid raft-associated molecules (i.e., CD20 and CD52) but not those against the non-raftassociated molecule CD33 further supported our proposed mechanism of action. Our results argue that concomitant medications must be adjusted carefully to achieve optimal antitumor effects with monoclonal antibodies. Cancer Res; 70(11); 4292-6. ©2010 AACR.

2354 Poster Board II-331 A defect in the programmed cell death, apoptosis, is implemented in the pathogenesis of CLL. About ten years ago, it became evident that patients with CLL can be divided into those with an indolent course of the leukaemia and those which suffer from a more aggressive disease, typically requiring frequent chemotherapy and ultimately develop a chemotherapy-refractory state. The latter group of patients aberrantly express the T-cell associated protein ZAP-70. The object of this study was to identify the molecular differences underlying the pathogenesis of these two CLL subgroups. To study differences in the apoptotic program we used primary CLL cells derived from untreated ZAP-70 negative and positive patients. Here we show that the expression of ZAP-70 enhances the signals associated with the B-cell receptor (BCR) and recruits protein kinase C-beta (PKC-beta) into lipid raft domains only in patients with an aggressive variant of the disease. Subsequently, PKC-beta is activated and shuttles from the plasma membrane into the mitochondria. By using co-immunoprecipitation experiments and PKC-beta specific small molecule inhibitors we unravel that the anti-apoptotic protein Bcl-2 and its antagonistic BH3-protein Bim are putative substrates for PKC-beta. PKC-beta mediated phosphorylation of Bcl-2 augments its anti-apoptotic function by increasing its ability to sequester more pro-apoptotic Bim. In addition, the phosphorylation of Bim by PKC-beta leads to its proteasomal degradation. Therefore, high levels of phospho-Bcl-2 and low levels of Bim are a hallmark of ZAP-70 positive, aggressive CLL. Importantly, posttranscriptional modifications of Bcl-2 seem to outweigh the absolute expression of Bcl-2 with respect to the suppression of apoptosis. We demonstrate that these cells are strongly protected from chemotherapy-induced cytotoxic stress. Our data indicate that the constitutive activation of PKC-beta is directly involved in the apoptotic defect in ZAP-70 positive CLL. We finally show that targeting PKC-beta is an attractive approach to the treatment of CLL patients. Enzastaurin is a PKC-beta specific inhibitor and currently tested in clinical phase I/II trials for cancer patients. Our data demonstrate that this compound is highly active in CLL cells and augments the cytotoxic effects of standard chemotherapeutic drugs. Our results provide evidence that the constitutive activation of PKC-beta is directly implicated in the pathogenesis of aggressive CLL by altering the function of the apoptosis-regulating proteins Bcl-2 and Bim. These changes confer cells to a more anti-apoptotic state with aggressiveness of the disease. Targeting PKC-beta with small-molecule inhibitors like Enzastaurin might offer a new therapeutic strategy to control or even cure CLL. Disclosures: No relevant conflicts of interest to declare.