ALBERT

Description: 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. The use of methylprednisolone (Mp) is widespread in the treatment of B-CLL related concomitant auto-immune disorders. We have recently demonstrated that Zap-70+ B-CLL cells are more resistant to chemotherapy but more susceptible to apoptotic cell death by steroids than Zap-70 negative cells in vitro. We investigated the possible influence of Mp on the expression of Zap-70 in B-CLL cells. Mononuclear cells of 15 patients with B-CLL were isolated by Ficoll centrifugation and incubated with different concentrations of Mp (range 0.1–500 μM). After 24h and 48h, Zap-70 expression, phospho-ZAP-70 (Tyr 319 and Tyr 493) and apoptosis were evaluated by flow cytometry (indirect staining ZAP-70 and phospho-ZAP-70 and Annexin V-FITC/propidium iodide double staining for apoptosis) and Western blotting when appropriate. Student’s t-test was used for statistical analysis. Mp induces time and dose dependent apoptosis in B-CLL cells (LD50 48h=16.8 μM). After 24h and 48h of incubation at doses of 50 μM, Zap-70 expression decreased significantly (50 μM: p

Description: 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.

Description: 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.

Description: Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy which has an incidence of 40 children on average per year in the Netherlands and Belgium combined (population of 28 million). Despite the fact that almost all children achieve complete remission (CR), 30-40% of patients eventually relapse. Since survival after relapse is poor (35-40%), the overall survival (OS) of pediatric AML remains relatively low (~70%) compared to the increasing remission rates. The most effective strategy to improve the dismal outcome of AML patients is thus to prevent relapse. Over the last decades accumulating evidence is suggesting that AML develops in a hierarchical structure; originating from hematopoietic stem cells which are transformed to leukemia initiating cells, also referred to as leukemic stem cells (LSC). LSC possess self-renewal capacity and are more resistant to therapy. Therefore, LSC are supposed to be responsible for outgrowth of both the initial leukemia and the relapse. This holds true for adult AML, where the frequency of LSC at diagnosis has shown to be of importance for clinical outcome. However, while it has been shown that a high number of immature cells (CD34+/CD38-/CD45-/low) associates with an increased risk of relapse in pediatric AML, relatively little is known about the prognostic impact of LSC within this compartment. RAEB-t. Flow-cytometric LSC characterization was performed on either bone marrow (BM) or peripheral blood (PB) from AML patients collected at diagnosis. Purified white blood cells (WBC) were obtained after lysing the red blood cells using lysing solution (Pharm Lyse or FACSLysing, BD Biosciences) and were subsequently incubated with monoclonal antibody combinations and analyzed using an 8-color flow cytometer approach. LSC were defined as CD34+ CD38- cells with aberrant expression of CD123, CD7, CD56 or CD2. Results: Data were available from 103 patients and patient characteristics are listed in Table 1. In this cohort relapse-free survival (RFS) was 59.4%. Fortunately 56.4% of relapsed patients achieved a second CR and OS was 85.4%. LSC-load is defined as % of aberrant CD34+ CD38- cells within the CD34+ compartment. In our pediatric cohort, 17 patients (16.5%) had no expression of CD34 on blast cells at all (CD34null) and consequently no aberrant CD34+ CD38- LSC could be detected. Absence of CD34 has often been associated with good prognosis in literature. In our cohort relapse-rate seemed to follow this trend (4 out of the 17 CD34null patients (23.5%) vs. 34 out of the 83 CD34positive patients (41%)) but did not differ significantly (Plogrank = 0.196). ROC curve analysis showed that a cut-off of 17.2% LSC at diagnosis was associated with the occurrence of developing relapse with a specificity of 92%. Kaplan-Meier survival analysis, as depicted in Figure 1, showed a significant association between a high LSC-load and impaired RFS (34% relapses in LSClow vs. 64% relapses in LSChigh) (Plogrank= 0.027). Univariate analysis showed that next to LSC percentage, FLT3 mutation status and WBC count were significantly associated with RFS. After multivariate adjustment (taking into account cytogenetic risk groups and mutational status) LSC frequency was the only independent predictor of relapse or RFS (HR 2.3, 95% CI 1.1-4.8, p=0.032). Conclusion: Our results confirm data from adults and reveal that the frequency of LSC at diagnosis in pediatric AML patients can distinguish patients more likely to fail current treatment regimens as these patients develop significantly more relapses). Identifying this patient group creates opportunities for more personalized medicine and the development of therapies directed against LSC, sparing normal hematopoietic stem cells. Disclosures Kaspers: Janssen-Cilag: Research Funding. Cloos:Takeda: Honoraria.

Description: In B-cell chronic lymphocytic leukemia (B-CLL) the mutation status of the immunoglobulin variable heavy chain gene (VH) is known as a prognostic factor. We have investigated if specific VH-gene usage is of additional prognostic importance regarding survival. Peripheral blood samples from 147 B-CLL patients were analysed for VH usage. Recombinations occurring in at least 5% of cases were studied in depth. The most frequently used VH-gene segments were VH1-69 (10.9%), VH3-7 (7.5%), VH3-30 (6.8%), VH4-34 (6.8%), VH3-21 (6.1%), VH3-23 (6.1%), and VH3-33 (5.4%). The VH gene usage was compared with mutation status, cytogenetic abnormalities and survival. Comparison with age matched controls reveals the restricted VH gene usage in B-CLL. VH gene usage showed a distinct prognostic value (p=0.01) when the patients using VH genes with bad prognosis were grouped together (VH1-69, VH3-21, VH3-23 and VH3-33; 43% of these patients died) and were compared with the patients using VH genes with a relatively good prognosis (VH3-7, VH3-30 and VH4-34; mortality rate of only 13%). The prognostic significance holds true when all patients were included (p=0.03). Also the mutation status (p=0.04) and cytogenetics (p=0.03) showed a prognostic significance. The VH gene usage did not correlate with mutation status, nor with cytogenetics. On the contrary mutation status and cytogenetics were strongly correlated (p

Description: Key Points LIN28B is overexpressed in about half of juvenile myelomonocytic leukemia patients and defines a novel fetal-like disease subgroup. LIN28B expression is correlated with high fetal hemoglobin levels and the absence of monosomy 7.

Description: Introduction Zap70 is superior to mutation status as a prognostic indicator in B-CLL, but whether it is causally implicated or just associated with progressive disease is unclear. Zap-70 transduces the TCR signal through the CD3 zeta-unit in T cells, but although it is important in preB development, it has no known function in mature B-cells. Zap-70 positive B-CLL show enhanced B cell receptor mediated Syk-phosphorylation upon surface IgM (sIgM) crosslinking, simulating antigen driven B cell proliferation. This may increase the proliferative drive in Zap-70+ B-CLL compared to Zap-70- leading to a higher risk of secondary chromosomal damage. Zap-70 was recently implied in Erk activation via CXCL-12/CXCR4 in T-cells and this is enhanced by cell adhesion through beta-1 integrins that may influence P-Erk phosphatases. Since both CXCL12/CXCR-4 and integrins influence B-CLL survival in vitro, we investigated the duration of downstream Erk activation in Zap-70 + versus Zap-70 B-CLL after CXCL-12 stimulation in the presence and absence of integrin binding by fibronectin. Materials/methods Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-gradient. Zap-70 was measured by flow cytometry (Upstate Ab), and PCR. B-CLL cells from 6 patients were stimulated with 100 ng/ml CXCL-12 after preincubation in serum free RPMI for 2 hrs at 37°in fibronectin or BSA coated plates and in suspension culture. After CXCL-12 addition supernatant and adherent cells were separately lysed at 0, 2 and 10 minutes and studied by Western blot for total Erk, Akt, P-Erk and P-Akt. Parallel cultures were extended for 24 hours and apoptosis of supernatant versus adherent cells was performed by AnnexinV-PI staining and flow cytometry. Results Erk-phosphorylation was markedly stimulated by CXCL-12 after 2 minutes, both in supernatant and adherent fractions of fibronectin and BSA coated plates compared to suspension cultures. This stimulation decreased in Zap-70- B-CLL after 10 minutes but remained high in Zap-70+ B-CLL particularly in the fibronectin adherent fraction. These cells were also protected from spontaneous apoptosis at 24 hours when compared to supernatant Zap70+ and Zap-70- cells. Conclusion CXCL-12/CXCR4 ligation leads to prolonged Erk-phosphorylation in Zap-70+ but not in Zap70- B-CLL cells. Prolonged Erk-activation is required for nuclear translocation and effective signal transduction along this way. This effect is enhanced by concomitant fibronectin engagement of integrins, which may give Zap-70+ B-CLL cells a proliferative advantage upon homing in lymphoid tissues.

Description: Introduction: Despite the use of current risk classification in children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), a substantial proportion of so-called standard risk patients will experience a hematological relapse. Detection of DNA copy number abnormalities in BCP-ALLs has revealed additional genetic alterations, some of which are associated with outcome and may be included in future stratification strategies. Materials and methods: Using array-comparative genomic hybridization in a selected cohort of 70 intermediate risk pediatric BCP-ALLs, we characterized a recurrent RAG-mediated deletion of the CD200 and BTLA genes in 10% of patients. A breakpoint-specific PCR assay was designed and used to screen an independent non-selected cohort of 1154 genetically well-characterized BCP-ALLs uniformly treated according to the BFM-based EORTC-58951 protocol. Results: CD200/BTLA deletions were identified in 56 patients of the non-selected cohort (4.8%). Survival analysis revealed that CD200/BTLA deletions are associated with an inferior 8-year event free survival (EFS) of 70.2% ± 1.2% for patients with the deletions versus 83.5% ± 6.4% for non-deleted cases (HR 2.02; 95% CI 1.23-3.32; p=0.005) in the complete cohort of 1154 BCP-ALL patients. We observed a strong association between CD200/BTLA deletions and ETV6-RUNX1 positive leukemias (Table 1). The presence of ETV6-RUNX1 is a good prognostic marker in BCP-ALL and CD200/BTLA deletions did not affect prognosis within this genetic subtype. However and most notably, CD200/BTLA deletions were also identified in patients without any known genetic lesion, who are classified as having an intermediate outcome and belong to the intermediate-risk genetic group (defined in Table 1). Within this genetic group an inferior 8-year EFS rate of 33.3% (95% CI 7.8%-62.3%) was observed for patients with the deletions versus 76.2% (95% CI 71.0%-80.6%) for non-deleted cases (HR 4.00; 99% CI 1.34-11.93; p