Background: The B-cell receptor (BCR) has a key role in the cross-talk between chronic lymphocytic leukaemia (CLL) cells and the tissue microenvironment, which favours disease progression by promoting proliferation and drug resistance. In vitro studies on downstream signalling and functional effects of CLL BCR ligation often report contradictory results, in part owing to the lack of a standardised stimulation protocol. Our aim was to define a biologically relevant and robust in vitro stimulation method with regard to cellular phenotypic and transcriptional responses.Methods: We evaluated mRNA (FOS, MYC, LPL) and protein (CD54, CD19, CD62L, CD184) expression of genes modulated by BCR triggering in immunoglobulin heavy-chain variable region genes (IGHV)-mutated and -unmutated CLL cells, after stimulation using soluble or immobilised anti-IgM antibodies from different suppliers.Results: The effect of BCR stimulation on gene and protein expression was comparable in all CLL patients, irrespective of IGHV mutation status. However, immobilised anti-IgM stimulation elicited clear and robust changes in gene and protein expression, whereas the response to soluble anti-IgM was far less obvious.Conclusions: These data indicate that the method of BCR stimulation is of major importance regarding responsiveness of CLL cells in the context of the tumour microenvironment, whereas genetic differences in the BCR pathway are less critical.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and is characterized by a heterogeneous clinical course. This variability in clinical course has spiked the search for prognostic markers able to predict patient evolution at the moment of diagnosis. Markers demonstrated to be of value are the mutation status of the immunoglobulin heavy chain variable region genes (IGHV) and lipoprotein lipase (LPL) expression. High LPL mRNA expression has been associated with short treatment free (TFS) and decreased overall survival (OS) in CLL. The LPL SNPs rs301 (T<C), rs328 (C<G) and rs13702 (T<C) have been associated with various metabolic disorders, but the association with CLL evolution is unknown. Here, in a cohort of 248 patients, we show that patients with the LPL SNP rs13702 wild-type T/T genotype had significantly shorter OS than patients with C/C and T/C genotypes (median time until CLL related death: 90 and 156 months respectively, p=0.008). The same was observed for LPL SNP rs301 (median time until CLL related death T/T: 102 and C/C, T/C: 144 months, p=0.03). Both SNPs rs301 and rs13702 were significantly associated with each other and notably, no association was found between IGHV status and presence of the SNP genotypes, indicating that these LPL SNPs are reliable prognostic markers that could add extra prognostic and predictive information to classical markers and help to improve the management of CLL.

The process of B-cell development is characterized by the activation of the unfolded protein response. Under certain circumstances, the unfolded protein response can be manipulated in a cell death-inducing way. Therefore, tackling the unfolded protein response might be an attractive strategy in the treatment of diffuse large B-cell lymphomas. Our research showed more basal unfolded protein response activity and differences in the inducibility of ER stress in activated B-cell versus germinal center cell lines. Moreover, the diffuse large B cell lymphoma patient data revealed that the glucose-regulated protein 94 is new potential discriminator for diffuse large B cell lymphoma.

Activation of the adaptive Ire1-XBP1 pathway has been identified in many solid tumors and hematologic malignancies, including multiple myeloma (MM). Here, we report the identification of STF-083010, a novel small-molecule inhibitor of Ire1. STF-083010 inhibited Ire1 endonuclease activity, without affecting its kinase activity, after endoplasmic reticulum stress both in vitro and in vivo. Treatment with STF-083010 showed significant antimyeloma activity in model human MM xenografts. Similarly, STF-083010 was preferentially toxic to freshly isolated human CD138(+) MM cells compared with other similarly isolated cell populations. The identification of this novel Ire1 inhibitor supports the hypothesis that the Ire1-XBP1 axis is a promising target for anticancer therapy, especially in the context of MM.

Normal B cell development is tightly regulated by transcription factors and is characterized by the formation of the B-cell receptor. When the regulation and activation of B cell differentiation is disrupted, lymphomas and leukemias can occur. These lymphoid malignancies often resemble normal stages of the B cell differentiation. During normal B cell development a precursor B cell will ultimately form a terminal differentiated lymphocyte or plasma cell. These terminal differentiated B cells require massive increase in their biosynthetic capacity to synthesize thousands of antibodies per second for secretion. For this purpose, these cells rely on a highly developed endoplasmic reticulum (ER), the compartment where proteins are assembled and modified into functional antibodies. However, upon perturbations in ER function a stress pathway can be activated, named the unfolded protein response (UPR). In the first place, the UPR is a cytoprotective signaling and several studies have demonstrated already that activation of the UPR might have a crucial role in cancer. Until now, little is known about the role of the UPR in the germinal center (GC)-derived lymphomas since most investigations were performed on healthy B cells and multiple myeloma. Therefore, the initial aim of this doctoral work was to evaluate the expression levels of baseline and inducible UPR-related markers in DLBCL cell lines and in a plasma cell line. Besides its protective role, the UPR may balance in the direction of cell death upon severe and prolonged stress. Manipulation of the UPR may be used as an alternative cell death mechanism since conventional treatments frequently activate the classical cell death pathways, like the intrinsic and extrinsic cell death program. Therefore, this cell death strategy is used on CLL patient samples. To accomplish this goal, the anticancer activities of two plant-derived compounds, xanthohumol (isolated from hop, Humulus lupulus L.) and withaferin A (isolated from Withania somnifera), were investigated. In conclusion, our results provide a scientific basis for further investigations of the involvement of the UPR in DLBCL. Moreover, targeting the ER may be an interesting therapeutic strategy for CLL and other lymphoid malignancies, and for further development of new therapeutic agents.

We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G(2)/M phase and stimulated an accumulation of the cells in the sub-G(0) phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-x(L). Pretreatment with the pancaspase inhibitor Z-VAD-FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78 kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib.

Chronic lymphocytic leukaemia remains the most common adult leukaemia in Western countries. In this review we tried to give an overview of the important prognostic markers and the developments in this field. We also reflected about the accumulative character of the disease and the influence of the micro-environment on the CLL cell.

BACKGROUND:Chronic lymphocytic leukemia (CLL) is an incurable disease with a natural history of increasing resistance to chemotherapy. A novel approach to overcome chemotherapy resistance may be targeting the endoplasmic reticulum (ER). PATIENTS AND METHODS:The involvement of the unfolded protein response (UPR) in the cell killing effect of xanthohumol (X) was examined in 18 patient samples. RESULTS:X-induced apoptosis of CLL cells was accompanied by the induction of glucose-regulated protein of 78 kDa (GRP78) and heat-shock protein of 70 kDa (Hsp70) protein levels and by sustained phosphorylation of the eukaryotic translation initiation factor 2 (eIF2alpha), suggesting the involvement of the ER stress transducer, the double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK). The X-box-binding protein 1 (XBP1) mRNA was spliced but no clear activation of activating transcription factor 6 (ATF6) was observed. The proapoptotic outcome was further demonstrated by the up-regulation of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), down-regulation of myeloid cell leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2), cleavage of poly-(ADP)-ribose polymerase (PARP) and processing of caspase-3, -4 and -9. Furthermore, X showed proteasome inhibitory activity. CONCLUSION:X stimulates the proapoptotic arm of the UPR in ex vivo CLL cells, suggesting that ER stress may play an important role during X-induced apoptosis.

In recent years, a lot of attention has been paid to phyto-chemicals such as flavonoids with anticancer effects since they may interact with cellular signaling pathways controlling proliferation, differentiation, apoptosis and invasion. Invasion is the hallmark of malignancy, and the search for anti-invasive agents remains a challenge. A number of studies reported a promising role for xanthohumol (X) as a chemopreventive agent, since it can modulate the carcinogen metabolism and act by cytotoxic/-static mechanisms. Recently, X was investigated for its anti-invasive activity on human breast cancer cell lines and shown to inhibit the invasion of breast cancer cells in different invasion assays. One of the possible mechanisms of the anti-invasive effect of X includes the involvement of the E-cadherin/catenin invasion-suppressor complex. Another mechanism by which X influences the invasive behavior is by reducing the number of invasive cells (i.e. by promoting cell death). Recent data demonstrate that X induces a specific stress reaction (i.e. endoplasmic reticulum stress) followed by the unfolded protein response and programmed cell death (apoptosis). In this overview, we highlight the effect of X on invasion and survival of breast cancer cells and the mechanisms associated with these effects.

There is resurgent interest in glucocorticoids (GCs) in the treatment of poor prognosis chronic lymphocytic leukemia (CLL). Little is known however on how GCs induce apoptosis in CLL. Methylprednisolone (MP) induces apoptosis in ZAP-70 positive CLL more readily than in ZAP-70 negative CLL, which is in contrast to the effects of radiation and chemotherapy. The increased GC sensitivity of ZAP-70+ CLL was studied in relation to the expression status of ZAP-70 and the related signal transducing tyrosine kinase SYK. Both ZAP-70 and SYK were downregulated by GC treatment. Moreover, SYK was dephosphorylated by the phosphatase PTP1B of which the expression and translation levels were induced by GCs. Inhibition of PTP1B successfully restored ZAP-70 expression and SYK phosphorylation but did not interfere with GC-induced apoptosis. Therefore, the downregulation of ZAP-70 and P-SYK per se during treatment with GCs is not sufficient to induce apoptosis, and different mechanisms must therefore be responsible for the increased steroid sensitivity of ZAP-70+ CLL.

7-Hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3′,2′:4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1) is a newly identified intestinal microbial metabolite from the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Although the mutagenic potential of the endogenous N-hydroxy PhIP derivate has been reported, the risks associated with PhIP-M1 have not yet been explored. In this work, the cytotoxic and genotoxic effects originating from PhIP-M1 were assessed in the epithelial intestinal Caco-2 cell line. PhIP-M1 significantly decreased in a time- and dose-dependent manner mitochondrial dehydrogenase activity and protein synthesis, with IC50 values of, respectively, 180±39.4 and 173±20.3μM after 24h, and 33.8±3.5 and 37.3±10.9μM after 72h. Apoptosis within the concentration ranges of cytotoxicity was confirmed by morphological examination, DAPI nuclear staining and annexin V staining. PhIP-M1 provoked cell cycle arrest, characterized by a significant increase in the number of nucleoids in the G2/M phase. A dose-dependent increase in DNA damage, as quantified by the alkaline comet assay, was observed after 3h in the 50–200μM range. Because these PhIP-M1-induced genomic and cellular events may contribute to the carcinogenicity of PhIP, the potency of the colon microbiota to bioactivate PhIP must be included in future risk assessments.

In eukaryotic cells, the endoplasmic reticulum (ER) is the principal site for the folding and maturation of transmembrane, secretory and ER-resident proteins. Functions of the ER are affected by various intracellular and extracellular stimuli, which include inhibition of glycosylation, reduction of disulfide bonds, calcium depletion from the ER lumen, impairment of protein transport to the Golgi, and expression of mutated proteins in the ER. Under ER stress, unfolded/misfolded proteins accumulate in the ER lumen, which induces conflicting cellular activities: survival and apoptosis. To cope with this stress, cells activate intracellular signalling pathways, such as the unfolded protein response (UPR) and the ER-associated degradation (ERAD). However, under conditions of severe ER stress or when the UPR has been compromised, the cell may be incapable of maintaining ER homeostasis, which may eventually activate programmed cell death (PCD) pathways. Clinical data support the potential of drugs that inhibit the normal functions and homeostasis of the ER and the proteasome in treatment of malignancies like cancer. It is therefore reasonable to assume that manipulation of ER stress might enhance the efficacy of chemotherapeutic drugs and provide new anticancer targets like the ER and the proteasome.

Glucocorticoids (GCs) are often implemented in the treatment regimens of B-cell Chronic Lymphocytic Leukemia (B-CLL). Apart from caspase-involvement and downregulation of Lyn, the exact mechanisms of action in apoptosis induction in B-CLL remain uncertain. We studied methylprednisolone (MP)-induced apoptosis in B-CLL and found a greater sensitivity of ZAP−70+ B-CLL cells for MP-induced cell death, which was accompanied by a glucocorticoid receptor-dependent decrease of ZAP-70 expression. Micro-array data correlated this downregulation of ZAP-70 with an upregulation of the phosphatase PTP1B (Protein Tyrosine Phosphatase 1B). This observation was confirmed on genomic and protein level by RT-qPCR and Western blotting. Next, functional assays with the phosphatase-inhibitor ortho sodium vanadate and with the PTP1B-inhibitor 3-(3,5-Dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonicacid-(−4-(thiazol-2-ylsulfamyl)-phenyl)-amide were performed. Both drugs were able to inhibit the GC-induced ZAP-70 downregulation, which is suggestive for the role of PTP1B. In order to explore this further, we studied the effect of MP on the phosphorylation of Syk by Western blotting and flow cytometry. We did not take phosphorylated ZAP-70 into account as phosphorylation of ZAP-70 is very unclear in CLL cells. We observed GC-induced dephosphorylation of Syk (tyrosine residues 352 and 526), associated with a decrease of total Syk. The kinetics were completely in accordance with those of the ZAP-70 downregulation, occurring only after 12 hours after the start of treatment. Next, we performed immunoprecipitation experiments to investigate the reciprocal influence of Syk and ZAP-70 and found co-immunoprecipitation of Syk, ZAP-70 and hsp90, suggesting a close relation of ZAP-70 and Syk in performing their tasks. In spite of the unclear status of ZAP-70 phosphorylation, ZAP-70 positive B-CLL cells allow for more effective IgM-signaling than their ZAP-70 negative counterparts, probably by blocking inhibitors of signalling through Syk. Moreover, ZAP-70 positive B-CLL cells undergo stronger and prolonged BCR-induced phosphorylation of tyrosine residues 352 and 526 of Syk, which are already prominent in unstimulated conditions. The kinetics and concentration overlapping downregulation of ZAP-70 and dephosphorylation of Syk after MP treatment suggests a relation between these two protein tyrosine kinases in performing their tasks. This was further strengthened by the observation of a co-immunoprecipitation of ZAP-70, Syk and hsp90. In conclusion, in vitro GC treatment of ZAP-70+ B-CLL cells is associated with a downregulation of ZAP-70 and a dephosphorylation of Syk tyrosines 352 and 526 via a GR-dependent way. The upregulation of PTP1B at both genomic and protein level, and the results of the functional assays, are suggestive for a role of this phosphatase during treatment with GCs.

B-Chronic Lymphocytic Leukemia (B-CLL) is incurable by current methods because of increasing resistance to chemotherapy. Therefore, new options are needed for the treatment of CLL. The proteasome-inhibitor bortezomib was described to promote apoptosis in CLL cells and to induce endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in refractory multiple myeloma. The UPR is mainly a self-protective mechanism activated when protein folding is disrupted and misfolded proteins are accumulating in the ER. However, sustained ER stress eventually leads to cell death. Three major ER sensors are involved during UPR, namely ATF6, PERK and IRE1. In inactivated state, they are bound to the ER-chaperone BiP/GRP78. Upon ER stress, BiP is released from the luminal domain of the ER stress transducers resulting in their activation and downstream signaling. Pro-survival signals are delivered by IRE1 (splicing of XBP1) and ATF6 (cleavage), while pro-apoptotic signals are generated by PERK (upregulation of CHOP). Previously, we demonstrated that the xanthohumol (X) is able to kill B-CLL cells in a dose- and time-dependent way as evidenced by PARP cleavage and Annexin V staining (Lust et al., 2005). In this study, we first demonstrated that X induces apoptosis of B-CLL cells in part via activation of ER stress and the UPR and identified the associated molecular markers. Treatment of freshly isolated B-CLL cells with X, stimulated the expression BiP and Hsp70 (suggestive for ER stress), the phosphorylation of eIF2a (suggestive for PERK activation), and the splicing of XBP1 mRNA (indicative for IRE activation). In contrast, ATF6 activation seemed not to be implicated since no cleaved ATF6 could be demonstrated. Induction of UPR was associated with a pro-apoptotic outcome evidenced by upregulation of CHOP, generation of ROS, downregulation of the anti-apoptotic proteins Mcl-1, Bcl-xL, Bcl-2, cleavage of PARP, and processing of caspase-3. Next, we showed that X inhibited the 20S proteasome activity in reticulocyte lysates but also functionally in B-CLL as demonstrated from the accumulation of polyubiquitin-conjugates under X treatment. The activation of UPR and influence on the anti-apoptotic proteins can mostly be explained by this proteasome inhibitory activity. In conclusion, we identified proteasome inhibitory capacities of xanthohumol in B-CLL in vitro. Proteasome-inhibition was accompanied by ER stress, UPR and apoptosis. Our results further suggest that tackling organelles like the proteasome and the ER is a valuable strategy in treatment of B-CLL.

Introduction Correct folding of new proteins is supervised in the endoplasic reticulum (ER) unfolded protein response (UPR). Misfolded proteins recruit the chaperone Grp78 that is thereby released from the transcription factors ATF6, IRE-1 leading to compensatory increase in Grp78, and PERK, leading to phosphorylation of eIF2α and block of further protein translation. UPR overload leads to ER stress and cell death. Targeting the endoplasmic reticulum (ER) is a new strategy explored in B-CLL. The hop-derived chalcone Xanthohumol (X) has been characterized as a ‘broad-spectrum’ cancer chemopreventive agent. Recently, we demonstrated that X induces dose- and time-dependent cell death of MCF7/6 breast cancer cells accompanied by ER stress. X induces apoptosis and cleavage of poly(ADP)-ribose-polymerase (PARP) in B-CLL in vitro. The present study investigates the branches of the UPR in relation to X induced apoptosis of B-CLL cells. Materials and methods. Lymphocytes were isolated by Lymphoprep from 15 patients with B-CLL after informed consent. CD19 positive cells were selected by EasySep positive selection kit. Apoptosis was assessed by flow-cytometry (AnnexinV-PI). Western Blotting was used for Grp78, ATF6, XBP1, phospho-eIF2a, eIF2a, ATF4, CHOP, phospho-IKK, IKK, PARP, caspase-9, -8, -7, -4, cleaved caspase-3, mcl-1, bcl-x L , bax, bak, and bid. NF-kB activity was assessed by EMSA. Quantitative RT-PCR was performed to analyze Grp78 mRNA levels. Bcl-2 protein level was detected by flow cytometry and reactive oxygen species (ROS) by fluorescence microscopy. Results and conclusion X induced an upregulation of Grp78 mRNA levels which was not translated in an increase in protein. X treatment stimulated a rapid and sustained phosphorylation of eIF2a, suggesting the involvement of PERK. In contrast, the ER-stress transducers ATF6 and IRE1 were not activated. X-induced ER stress was associated with strong induction of the pro-apoptotic protein CHOP and inhibition of the NF-kB pathway. Furthermore, the pro-apoptotic effect of X was accompanied by an accumulation of ROS, a downregulation of the anti-apoptotic proteins mcl-1, bcl-x L , bcl-2 and processing of caspase-3, -7 and -9.In conclusion, the chalcone X is capable of inducing cell death with down-regulation of bcl-2, mcl-1, bcl-xL, and activation of the caspase cascade. This is accompanied by ER-stress as evidenced by the upregulation of Grp78 mRNA levels, induction of a rapid and sustained phosphorylation of eIF2a, upregulation of CHOP, and inhibition of the NF-kB signaling.

Zap-70 is accepted as a surrogate marker for mutational status of immunoglobulin heavy-chain variable region genes in B-CLL. Whether Zap-70 is a functional target for treatment of the more aggressive CLL cells that express unmutated IgVH is still under investigation.