Gerold Meinhardt

CpG DNA increases primary malignant B cell expression of costimulatory molecules and target antigens.

Multiple factors, including expression of costimulatory molecules, antigen‐presenting molecules, and target antigens, likely impact the efficacy of antibody therapy and other approaches to the immunotherapy of B cell malignancy. Unmethylated CpG‐dinucleotides in select base contexts (“CpG motifs”) that resemble sequences found in bacterial DNA are potent immunostimulatory agents capable of inducing a complex immune response, including a strong B cell stimulus. We examined the effect of a potent human CpG oligonucleotide (CpG ODN 2006) on different types of primary human malignant B cells and reactive follicular hyperplasia. CpG oligodeoxynucleotide (CpG ODN), but not control (non‐CpG ODN), increased the expression of costimulatory molecules (CD40, CD80, CD86, CD54) on malignant B cells without altering the phenotype of B cells obtained from reactive follicular hyperplasia. CpG ODN also enhanced expression of class I and class II MHC in most samples. CD20 expression was increased in response to CpG ODN, most notably in B‐CLL and marginal zone lymphoma. An inverse correlation was found between baseline expression of CD20 and CD40 and their expression after exposure to CpG ODN, thus the most significant increase in expression of these molecules was found in those samples that had the lowest baseline levels. In conclusion, CpG ODN can lead to increasing expression of molecules involved in costimulation, antigen presentation, and as targets for antibody‐based therapy and deserve further evaluation as potential immunotherapeutic agents for B cell malignancy.

STAT3 is constitutively active in some patients with Polycythemia rubra vera

OBJECTIVE Polycythemia vera is a clonal stem cell disorder characterized by hyperproliferation of the erythroid, myeloid, and megakaryocytic lineages. While it has been shown that progenitor cells of P. vera patients are hypersensitive to several growth factors including erythropoietin, insulin-like growth factor-1, thrombopoietin, interleukin-3, and granulocyte/monocyte colony-stimulating factor, the molecular pathogenesis of this disease remains unknown. Growth factor hypersensitivity could be mediated by changes in signal transduction pathways. We therefore investigated a common downstream effector of cytokines, the signal transducers and activators of transcription (STATs). A constitutive activation of STAT factors could explain the increased proliferation of P. vera cells even in the absence of growth factor stimulation. METHODS Peripheral granulocytes from patients with P. vera and from healthy volunteers were assayed for STAT1, 3, and 5 DNA binding by electrophoretic mobility shift assay. RESULTS Four of 14 P. vera patients analyzed showed constitutive STAT3 DNA binding in unstimulated peripheral granulocytes, while none of the 17 healthy volunteers tested did. None of the subjects showed constitutive STAT1 or STAT5 activity. Western blotting demonstrated that, in the three patients, STAT3 is constitutively phosphorylated on Tyr 705, whereas it is unphosphorylated in the other patients and in controls. Interestingly, constitutive STAT3 activity did not correlate with the duration of disease or the treatment regimen. It was observed in a recently diagnosed patient and in two patients treated only with phlebotomy. CONCLUSION Our data suggest that constitutive phosphorylation and activation of STAT3 is not a secondary event induced by mutagenizing agents or by prolonged hyperproliferation of hematopoietic cells, but rather represents a primary molecular aberration. Constitutively active STAT3 may contribute to the growth factor hypersensitivity of P. vera cells.

Molecular pathogenesis of chronic lymphocytic leukemia: factors and signaling pathways regulating cell growth and survival

Abstract Chronic lymphocytic leukemia is a malignant disease characterized by clonal expansion of relatively mature B-lymphocytes with a high percentage of cells arrested in the nonproliferative G0/G1 cell cycle phase. Possibly reflecting the clinical heterogeneity observed in patients, various signaling pathways may become affected during the initiation and course of this disease. This review discusses frequent alterations concerning proliferative, differentiation-inducing, and apoptotic pathways elucidated in the recent years that have improved our understanding of this disease.

Modulation of malignant B cell activation and apoptosis by bcl‐2 antisense ODN and immunostimulatory CpG ODN

Inhibition of bcl‐2 expression by antisense oligodeoxynucleotides (ODN) might render bcl‐2 overexpressing malignant B cells more susceptible to chemotherapy. ODN containing unmethylated CG dinucleotides (CpG) are known to activate B cells. We studied the effects of two bcl‐2 antisense ODN, with (G3139) or without CG dinucleotides (NOV 2009) within the sequence, and the effects of a nonantisense, CpG‐containing ODN (ODN 2006) on activation and apoptosis of malignant B cell lines and primary B‐CLL cells. Without cationic lipids, no antisense‐mediated inhibition of bcl‐2 synthesis was achieved with G3139 and NOV 2009. Instead, G3139, but not NOV 2009, induced similar changes as ODN 2006 in proliferation, expression of costimulatory and antigen‐presenting molecules, as well as in bcl‐2 and bcl‐xL levels of primary B‐CLL cells. G3139 and ODN 2006 inhibited in vitro, spontaneous apoptosis in B‐CLL cells of patients with high serum thymidine kinase activity (s‐TK, marker for proliferative activity of malignant B cells), whereas in patients with low s‐TK activity, apoptosis was induced. In conclusion, our results suggest that modulation of malignant B cell apoptosis by G3139 depends on its immunostimulatory properties rather than on antisense‐mediated reduction of bcl‐2 expression. Immunostimulatory CpG ODN may have a therapeutic potential in patients with B‐CLL, especially those with low s‐TK activity.

First clinical experience with simvastatin to overcome drug resistance in refractory multiple myeloma.

In vitro statins overcome cell adhesion‐mediated drug resistance at non‐toxic concentrations that are achievable in humans by standard dose simvastatin. A pilot phase‐II trial was initiated to determine feasibility and antimyeloma efficacy. In six myeloma patients refractory to two cycles of bortezomib or bendamustine simvastatin was concomitantly administered during further two cycles. The therapy was well tolerated without grade 3/4 toxicity. Intrapatient (cycles I/II vs. III/IV) and interpatient comparison (vs. ten patients without simvastatin) showed reduction of drug resistance by inhibition of HMG‐CoA‐reductase. In summary, this is the first phase II experience to study antimyeloma activity of statins in humans.

Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis

Activation of PKC with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in human U937 myeloid leukemia cells is associated with induction of adherence, followed by monocytic differentiation and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these effects about 25% of U937 cells accumulated in an apoptotic subG1 phase after TPA treatment. The appearance of these apoptotic suspension cells was detectable throughout the time course of the culture and was independent of TPA concentrations between 0.5 and 500 nM. Experiments with cells synchronized by centrifugal elutriation revealed dominant susceptibility of G1-phase cells to TPA-mediated apoptosis. While adherent cells expressed differentiation markers including the integrin CD11c, this effect was less pronounced in the TPA-treated suspension fraction. Moreover, previous work has demonstrated cell cycle arrest in differentiating U937 cells. Accordingly, PKC activation by TPA treatment was associated with a significant expression of the cdk/cyclin inhibitor p21WAF/CIP/sdi-1 in the adherent population and subsequent G0/G1 cell cycle arrest. In contrast, suspension cells failed to induce significant levels of p21WAF/CIP/sdi-1 after TPA stimulation. Immunoblotting experiments demonstrated no difference in the expression of the pro-apoptotic factors Bax, Bad, and Bak in either control U937 and TPA-treated adherent or suspension cells, respectively. However, anti-apoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 were significantly induced in the adherent population whereas no induction was detectable in the suspension cells. In this context, incubation with the caspase-3/caspase-7 specific tetrapeptide inhibitor DEVD prior to TPA treatment prevented an accumulation of cells in subG1, respectively, demonstrating an involvement of these caspases. Taken together, these data suggest that PKC activation can relay distinct signaling pathways such as induction of adherence coupled with monocytic differentiation and growth arrest, or induction of caspase-mediated apoptosis coupled with the failure to adhere and to differentiate. Cell Death and Differentiation (2000) 7, 795–803

Evidence for cell adhesion-mediated drug resistance of multiple myeloma cells in vivo

BACKGROUND/AIMS Multiple myeloma is an incurable disease and patients eventually die of disease progression due to drug resistance. VLA-4 (very late antigen 4), VCAM (vascular adhesion molecule), LFA-1 (leukocyte function-associ-ated antigen 1), and ICAM-1 (intercellular adhesion molecule 1)mediated adhesion of myeloma cells to bone marrow stromal cells induces primary multidrug resistance in vitro. Based on these preclinical data we hypothesized that myeloma cells with strong adhesion due to strong expression of adhesion molecules on the cell surface are selected by chemotherapy in patients. To prove this hypothesis we determined the expression levels of adhesion molecules in 31 multiple myeloma patients by flow cytometry. METHODS A 3-color stain with CD38, CD138 and antibodies against VLA-4, ICAM-1, LFA-1, and VCAM was performed. The patients were either at diagnosis (chemo-naive; n=17) or at relapse (pre-treated; n=15). Furthermore, the response to the next chemotherapy of chemo-naive patients was correlated with the expression levels of adhesion molecules. RESULTS ICAM-1, VLA-4, and VCAM expression was higher in pre-treated patients than in chemo-naive patients and the ex-pression levels increased with the number of chemotherapy regimens. Primarily multidrug-resistant patients had signifi-cantly higher expression levels of VLA-4 and ICAM-1 than responders. CONCLUSION This study suggests that multiple myeloma cells expressing high levels of VLA-4 and ICAM-1 are drug resistant and that such a subpopulation of cells is selected by chemotherapy.

Protein kinase C activation modulates pro- and anti-apoptotic signaling pathways

Activation of protein kinase C (PKC) by TPA in human U937 myeloid leukemia cells is associated with induction of adherence, differentiation, and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these differentiating cells about 25% of U937 cells accumulated in the subG1 phase after TPA treatment. This effect proved to be phorbol ester-specific, since other compounds such as retinoic acid or vitamin D3 failed to induce apoptosis in conjunction with differentiation. Only a specific inhibitor of PKC, GF109203X, but not the broad-spectrum kinase inhibitor staurosporine or a tyrosine kinase inhibitor genistein could reverse the induction of apoptosis. Bryostatin-1, another specific PKC activator with distinct biochemical activity failed to induce apoptosis. Moreover, bryostatin-1 completely abolished the induction of apoptosis in U937 cells even if added 8 hours after TPA treatment. Apart from apoptosis induced by various chemotherapeutic drugs, TPA-related cell death is not mediated by an autocrine Fas-FasL loop and could not be prevented by a blocking antibody to the Fas receptor. However, a 75% reduction in the number of apoptotic cells after TPA stimulation was achieved by preincubation with a blocking antibody to the TNFalpha receptor. Tetrapeptide cleavage assays revealed a four-fold increase in the DEVD-cleavage activity in U937 cells compared to a three-fold increase in TUR cells. Immunoblotting demonstrated that TUR cells did not activate significant levels of caspase-3 or -7, whereas in U937 cells a 20-kDa cleavage product corresponding to activated caspase-3 was detectable after 3 d TPA exposure. Moreover, immunoblots revealed a strongly reduced expression of the adaptor molecule APAF-1, which is required for cytochrome c-dependent activation of caspase-9 and subsequently caspase-3. APAF-1 proved to be inducible after PKC activation with phorbol ester in U937, but not in TUR cells. Thus, APAF-1 expression may, at least in part, be regulated by PKC activity and reduced APAF-1 levels are associated with resistance to various inducers of apoptosis. Furthermore, TPA exposure of U937 cells is associated with increased levels of the pro-apoptotic proteins Bak and Bcl-xs, whereas simultaneously a decline in the Bcl-2 expression was noticable.

Treosulfan is an effective inducer of cell death in myeloma cell lines and primary myeloma cells from patients

Summary. Multiple myeloma is a non‐curable haematological disease involving transformed plasma cells. High rates of complete remission can be achieved with autologous peripheral blood stem cell transplantation. Treosulfan is an alkylating substance that has been used in the treatment of ovarian carcinomas for many years. It has a favourable side‐effect profile even at high‐dose protocols. Thus, the objective of this study was to evaluate the effect of treosulfan on myeloma cells. The treatment of the myeloma cell lines, NCI‐H929 and U266, with treosulfan led to apoptosis in both cell lines in a dose‐ and time‐dependent manner. The induction of apoptosis was accompanied by cleavage of caspases ‐3 and ‐9 as well as downregulation of the antiapoptotic protein Mcl‐1 and upregulation of the inhibitor of cyclin‐dependent kinases, p21WAF1/CIP1. Furthermore, 100 µmol/l treosulfan was capable of inducing cell death in 63·6 ± 23·9% of primary myeloma cells, whereas treatment with the same concentration of melphalan showed 59·7 ± 26% cell death. These in vitro concentrations were at least 10‐fold lower than achievable plasma levels, even at conventional doses of treosulfan. Our results suggest that treosulfan might be an appropriate candidate for novel treatment protocols for patients with multiple myeloma.

Differential expression of c-myc, max and mxi1 in human myeloid leukemia cells during retrodifferentiation and cell death

Previous studies in human myeloid leukemia cells (HL-60, U-937, THP-1) suggested an involvement of the c-myc gene in the control of mutually exclusive pathways, such as retrodifferentiation and cell death. Treatment of U-937 cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) which is associated with the induction of a monocytic differentiation program and growth arrest, revealed an initial up-regulation of c-myc, c-max, and mxi1 mRNAs after 1-6 h. Thereafter expression of these genes significantly declined to barely detectable levels when the cells ceased to grow after 12-24 h of TPA treatment. Between 7 and 11 days of TPA-induced G0/G1 cell cycle arrest, expression of the c-max and mxi1 genes continuously increased up to 8-fold until 32 days and declined to control levels when the cells regained proliferative capacity by 36 days. In contrast, c-myc mRNAs remained down-regulated during periods of growth arrest and increased only during re-entry into the cell cycle after 36 days. This effect is consistent with a retrodifferentiation process, whereby previously differentiated cells revert back to the undifferentiated phenotype and re-enter the cell cycle. Different results were obtained during serum starvation-induced cell death of U-937 cells. After 48-72 h of serum-starvation, expression of the c-myc and c-max genes were significantly down-regulated by 4-fold and 3-fold, respectively, while there was little, if any, change in mxi1 mRNA levels. Analysis of cell death in serum-starved U-937 cells demonstrated progressively increasing DNA fragmentation reaching 45.4% +/- 0.9% after 72 h. Synchronization of proliferating U-937 cells throughout distinct phases of the cell cycle exhibited little, if any, change in c-myc, c-max and mxi1 mRNAs. Furthermore, like c-myc, c-max and mxi1 mRNA transcripts appeared to be regulated primarily by post-transcriptional mechanisms, and c-max and mxi1 half-lives exceeded 4 h in contrast to < 60 min for the c-myc gene. Taken together, these findings suggested differential regulation and inverse expression levels of c-myc compared to c-max and mxi1 during differentiation, retrodifferentiation and cell death.