Ingo Ringshausen

Cyclin-dependent kinase inhibitor Roscovitine induces apoptosis in chronic lymphocytic leukemia cells

A new class of cell cycle inhibitors is currently entering clinical trials. These drugs exert their activity by inhibition of cyclin-dependent kinases (cdk) and induce cell cycle arrest and apoptosis in cancer cells. Roscovitine, a cdk2-inhibitor that is in preclinical evaluation, induced apoptosis in B-CLL cells at doses that were not cytotoxic for normal human B cells. At 20 μM, Roscovitine induced apoptosis in 21 of 28 B-CLL samples and was equally effective in zap-70-positive or -negative samples. Caspase-3 was cleaved in B-CLL cells exposed to Roscovitine and the pancaspase inhibitor z.VAD.fmk-blocked Roscovitine-induced apoptosis. Expression of the proapoptotic protein Bak was increased and Bax cleavage and conformational change was observed in Roscovitine-treated B-CLL cells. Antiapoptotic proteins Mcl-1 and XIAP were downregulated, but the expression of Bcl-2 remained unchanged. In contrast to previous reports in cancer cell lines, Roscovitine treatment was not accompanied by nuclear accumulation of p53. Cyc202 (R-Roscovitine) is in early clinical trials in cancer patients. Given its powerful effects on zap-70-positive and -negative B-CLL cells, but not on normal lymphocytes, Roscovitine might be an attractive drug to be tested in this incurable disease.

Mammalian target of rapamycin inhibition induces cell cycle arrest in diffuse large B cell lymphoma (DLBCL) cells and sensitises DLBCL cells to rituximab

Diffuse large B‐cell lymphoma (DLBCL) is a common lymphoma entity. Although a significant amount of DLBCL patients can be cured with modern chemotherapeutic regimens, a substantial proportion of patients die because of progressive disease. Therefore, new therapeutic strategies are clearly needed. Inhibitors of mTOR [mammalian target of rapamycin (Rap)] represent a new class of antiproliferative drugs with applications as immunosuppressive and anticancer agents. Extensive safety data exist on the mTOR inhibitor RAD001, which is already approved as an immunosuppressant in organ transplant recipients. Rap and RAD001 inhibited cell cycle progression in DLBCL cells by inducing a G1 arrest without inducing apoptosis. Phosphorylation of the main targets of mTOR, p70 s6 kinase and 4‐EBP‐1 was reduced in cells cultured in the presence of RAD001. Cell cycle arrest was accompanied by reduced phosphorylation of the retinoblastoma protein (RB) as well as reduced expression of cyclin D3 and A in all cell lines. Although the effect of the chemotherapeutic agent vincristine (vin) was not enhanced by RAD001, rituximab‐induced cytotoxicity was augmented in the rituximab‐sensitive cell lines. mTOR inhibition is a promising therapeutic strategy in DLBCL by inducing a G1 arrest and augments rituximab‐induced cytotoxicity. Therefore, combination of these drugs might be an interesting new therapeutic approach in DLBCL patients.

Sorafenib induces cell death in chronic lymphocytic leukemia by translational downregulation of Mcl-1.

Chronic lymphocytic leukemia (CLL) has a high prevalence in western countries and remains incurable to date. Here, we provide evidence that the multikinase inhibitor sorafenib induces apoptosis in primary CLL cells. This strong pro-apoptotic effect is not restricted to any subgroup of patients, based on Binet stage and the expression of ZAP70 or CD38. Mechanistically, sorafenib-induced cell death is preceded by a rapid downregulation of Mcl-1 through the inhibition of protein translation. Subsequently, the cell intrinsic apoptotic pathway is activated, indicated by destabilization of the mitochondrial membrane potential and activation of caspase-3 and -9. In contrast to sorafenib, the monoclonal vascular epidermal growth factor (VEGF)-antibody bevacizumab failed to induce apoptosis in CLL cells, suggesting that sorafenib induces cell death irrespectively of VEGF signalling. Notably, although sorafenib inhibits phosphorylation of the Scr-kinase Lck, knock-down of Lck did not induce apoptosis in CLL cells. Of note, the pro-apoptotic effect of sorafenib is not restricted to cell-cycle arrested cells, but is also maintained in proliferating CLL cells. In addition, we provide evidence that sorafenib can overcome drug resistance in CLL cells protected by microenvironmental signals from stromal cells. Conclusively, sorafenib is highly active in CLL and may compose a new therapeutic option for patients who relapse after immunochemotherapy.

Recruitment of PKC-βII to lipid rafts mediates apoptosis-resistance in chronic lymphocytic leukemia expressing ZAP-70

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-βII (PKC-βII) into lipid raft domains. Subsequently, PKC-βII is activated and shuttles from the plasma membrane to the mitochondria. We unravel that the antiapoptotic protein Bcl-2 and its antagonistic BH3-protein BimEL are putative substrates for PKC-βII. PKC-βII-mediated phosphorylation of Bcl-2 augments its antiapoptotic function by increasing its ability to sequester more pro-apoptotic BimEL. In addition, the phosphorylation of BimEL by PKC-βII 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.

Inhibition of the proteasome induces cell cycle arrest and apoptosis in mantle cell lymphoma cells

Summary. Mantle cell lymphoma (MCL) is a distinctive non‐Hodgkins lymphoma subtype, characterized by overexpression of cyclin D1 as a consequence of the chromosomal translocation t(11;14)(q13;q32). MCL remains an incurable disease, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL, which is often associated with additional cytogenetic alterations, has an even worse prognosis and new treatment options are clearly needed. The present study investigated the effect of a specific proteasome inhibitor, lactacystin, on cell cycle progression and apoptosis in two lymphoma cell lines harbouring the t(11;14)(q13;q32) and additional cytogenetic alterations, including p53 mutation (NCEB) and p16 deletion (Granta 519). Granta cells were more susceptible to inhibition of the proteasome with respect to inhibition of proliferation and apoptosis induction. No changes were observed in the expression levels of the G1 regulatory molecules cyclin D1 and cdk4, but cell cycle arrest and apoptosis induction was accompanied by accumulation of the cdk inhibitor p21 in both cell lines. Increased p53 expression was only observed in Granta cells with wild‐type p53. Cleavage of procaspase‐3 and ‐9 was observed but cleavage of procaspase‐8 was not involved in apoptosis induction. The proapoptotic effect of lactacystin was reversed by pretreatment with the pancaspase inhibitor zVAD.fmk. Lactacystin was also effective in inducing apoptosis in lymphoma cells from MCL patients. We conclude that inhibition of the proteasome might be a promising therapeutic approach for this incurable disease.

Cycling B-CLL cells are highly susceptible to inhibition of the proteasome: involvement of p27, early D-type cyclins, Bax, and caspase-dependent and -independent pathways.

OBJECTIVE Although peripheral blood B-CLL cells are arrested in G0 phase of the cell cycle, a proliferating pool of cells in proliferation centers might be involved in disease progression. We have previously described an in vitro model of this proliferating pool of cells using B-CLL cells stimulated with immunostimulatory oligonucleotides (CpG-ODN) and interleukin-2. Lactacystin is a specific inhibitor of the proteasome and is a potent apoptosis inductor in resting peripheral B-CLL cells. In the present study, we investigated the effect of proteasome inhibition in proliferating B-CLL cells. METHODS The effect of proteasome inhibition was analyzed using thymidine incorporation, annexin V assays, and TUNEL staining. Immunoblots were performed to evaluate expression of proteins involved in cell cycle and apoptosis regulation. RESULTS Lactacystin blocked cell cycle progression in activated B-CLL cells and inhibited degradation of p27. Upregulation of cyclin D2 and D3 in activated B-CLL cells was inhibited while the expression of cdk2, cdk4, and cyclin E remained unchanged. Activated B-CLL cells were more susceptible to apoptosis induction as compared to resting B-CLL cells. Apoptosis induction was accompanied by cleavage of Bax, procaspase 8, procaspase 9, and procaspase 3. However, a broad-spectrum caspase inhibitor (z-VAD.fmk) only partially inhibited cell death although DNA degradation was completely inhibited. CONCLUSION Proteasome inhibition is highly effective in proliferating B-CLL cells and induces apoptosis using a caspase-dependent and -independent pathway.

Mammalian target of rapamycin (mTOR) inhibition in chronic lymphocytic B-cell leukemia: a new therapeutic option.

Chronic lymphocytic B-cell leukemia (B-CLL) is an incurable disease characterized by the accumulation of monoclonal mature B cells, although disease progression relies upon cycling B-CLL cells in proliferation centers in central lymph organs. Rapamycin and its analogs are immunosuppressant drugs that exert their activity by specific inhibition of the mammalian target of rapamycin (mTOR). mTOR inhibition induces cell cycle arrest not only in normal lymphocytes but also in malignant cells. Therefore, rapamycins have recently entered the field of cancer treatment. In the present review we discuss how progression through the cell cycle is regulated in B-CLL cells and how rapamycin and its analogs can be used as target therapies against proliferating B-CLL cells. We also focus on additional effects of rapamycin, such as targeting the interaction between malignant B cells and the microenvironment.

Immunotoxin BL22 induces apoptosis in mantle cell lymphoma (MCL) cells dependent on Bcl-2 expression

Mantle cell lymphoma (MCL) is an incurable mature B cell proliferation, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL has an even worse prognosis and new treatment options are clearly needed. We analysed the effects of BL22, an immunotoxin composed of the Fv portion of an anti‐ CD22 antibody fused to a 38‐kDa Pseudomonas exotoxin‐A fragment on four MCL cell lines as well as on primary cells of four MCL patients. Apoptosis induction by BL22 was much more pronounced in MCL cell lines with low Bcl‐2 expression (NCEB‐1, JeKo‐1 and JVM‐2) compared to Granta‐519 cells with high Bcl‐2 expression. While the expression of the antiapoptotic protein Mcl‐1 declined (NCEB‐1, Granta‐519), Bcl‐2 levels remained unchanged in Granta‐519 cells. However transfection of BCL2 cDNA into NCEB‐1, JeKo‐1 and JVM‐2 cells significantly reduced BL22‐mediated toxicity. Accordingly we examined the effects of Bcl‐2 inactivation in Granta‐519 cells using siRNA. Indeed, apoptosis induction was strongly enhanced in Granta‐519 cells with silenced Bcl‐2. Our results were confirmed in freshly isolated MCL‐cells from patients with leukaemic MCL. We conclude that Bcl‐2 expression is important for mediating resistance against the immunotoxin BL22 in MCL cells.

Antiapoptotic effect of interleukin-2 (IL-2) in B-CLL cells with low and high affinity IL-2 receptors.

Although B chronic lymphocytic leukemia (B-CLL) cells express the alpha chain of the interleukin-2 (IL-2) receptor CD25, little is known about the effect of IL-2 on apoptosis in B-CLL cells. We have shown previously that stimulation of B-CLL cells with a CpG-oligonucleotide induces IL-2 high affinity receptors. In our current work, we analyzed the effect of IL-2 on apoptosis in resting B-CLL cells and in our model of activated B-CLL cells (CD25 high cells). IL-2 had modest antiapoptotic activity in resting B-CLL cells. In contrast, IL-2 was much more potent to prevent apoptosis in activated cells. Prevention of cell death was also associated with the maintenance of the mitochondrial membrane potential. While only limited regulation of apoptosis controlling proteins was observed in resting B-CLL cells, IL-2 had strong effects on MCL-1, Bcl-xl, and survivin expression and inhibited Bax cleavage in CD25 high cells. Interestingly, expression of Bcl-2 was reduced. Addition of IL-2 to activated B-CLL cells caused rapid phosphorylation of Akt, while IL-2 failed to significantly phosphorylate Akt in resting B-CLL cells. Pharmacological inhibition of Akt by LY294002 restored sensitivity of activated B-CLL cells to fludarabine. IL-2 might be an important survival factor in activated B-CLL cells and might contribute to disease progression by upregulation of several critical antiapoptotic proteins.

Integration of innate into adaptive immune responses in ZAP-70–positive chronic lymphocytic leukemia

The crucial dependence of chronic lymphocytic leukemia (CLL) cells on signals derived from the B cell receptor (BCR) has encouraged the development of new inhibitors, which interfere with BCR signaling and demonstrate clinical benefits in nearly all patients. In addition, signaling through Toll-like receptor (TLR) 9 of the innate immune system has been shown to further contribute to the activation of CLL cells. However, responses to TLR9 engagement are not uniform, but diametrically opposed with cell death in some patients and cell proliferation in others. We now provide evidence that heterogeneous responses to TLR agonists are related to differences in the ability of CLL cells to activate the BCR-associated kinase Syk. Notably, expression of ZAP-70 appears to be of crucial importance for TLR9-mediated activation of Syk. We show that the activation of Syk provides an antiapoptotic signal, which is independent of Mcl-1, Bcl-2, and Bcl-XL, but related to the degradation of the proapoptotic Bim. Mechanistically, TLR9-mediated antiapoptotic signals in ZAP-70-positive CLL trigger secretion of immunoglobulin M, which then serves as (auto-) antigen for a prosurvival BCR signal. Thus, our data show that single activation of the innate immune receptor TLR9 is sufficient to fully engage BCR signaling in ZAP-70-positive CLL, protecting malignant cells from apoptosis. We conclude that the integration of TLR signaling into an adaptive immune response can further promote survival of CLL cells and may contribute to the unfavorable prognosis of ZAP-70-positive CLL.