Medhat Shehata

Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance

Marrow stromal cells (MSCs) provide important survival and drug resistance signals to chronic lymphocytic leukemia (CLL) cells, but current models to analyze CLL-MSC interactions are heterogeneous. Therefore, we tested different human and murine MSC lines and primary human MSCs for their ability to protect CLL cells from spontaneous and drug-induced apoptosis. Our results show that both human and murine MSCs are equally effective in protecting CLL cells from fludarabine-induced apoptosis. This protective effect was sustained over a wide range of CLL-MSC ratios (5:1 to 100:1), and the levels of protection were reproducible in 4 different laboratories. Human and murine MSCs also protected CLL cells from dexamethasone- and cyclophosphamide-induced apoptosis. This protection required cell-cell contact and was virtually absent when CLL cells were separated from the MSCs by micropore filters. Furthermore, MSCs maintained Mcl-1 and protected CLL cells from spontaneous and fludarabine-induced Mcl-1 and PARP cleavage. Collectively, these studies define common denominators for CLL cocultures with MSCs. They also provide a reliable, validated tool for future investigations into the mechanism of MSC-CLL cross talk and for drug testing in a more relevant fashion than the commonly used suspension cultures.

High expression of lipoprotein lipase in poor risk B-cell chronic lymphocytic leukemia.

We investigated the pattern of lipoprotein lipase (LPL) expression in B-cell chronic lymphocytic leukemia (B-CLL) and assessed its prognostic relevance. Expression of LPL mRNA as well as protein was highly restricted to leukemic B cells. The intensity of intracellular immunoreactivity of LPL was higher in samples of patients with unmutated immunoglobulin heavy-chain variable region genes (IGVH) compared to those with mutated IGVH genes. LPL mRNA levels in peripheral blood mononuclear cells (PBMNC) from 104 CLL patients differed by 1.5 orders of magnitude between cases with mutated (N=51) or unmutated (N=53) IGVH (median: 1.33 vs 45.22 compared to normal PBMNC). LPL expression correlated strongly with IGVH mutational status (R=0.614; P<0.0001). High LPL expression predicted unmutated IGVH status with an odds ratio of 25.90 (P<0.0001) and discriminated between mutated and unmutated cases in 87 of 104 patients (84%). LPL expression was higher in patients with poor risk cytogenetics. High LPL expression was associated with a shorter treatment-free survival (median 40 vs 96 months, P=0.001) and a trend for a shorter median overall survival (105 months vs not reached). Our data establish LPL as a prognostic marker and suggest functional consequences of LPL overexpression in patients with B-CLL.

Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process

The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patients prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold‐standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta‐associated protein (ZAP‐70) expression was associated with unmutated IgVH genes and ZAP‐70 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP‐70 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP‐70 protein expression levels in CLL cells and residual normal lymphocyte subsets. However, several factors introduce variability in the results reported from different laboratories; these factors include the anti‐ZAP‐70 antibody clone and conjugate, the staining procedure, the gating strategy, and the method of reporting the results. The need for standardization of the approach led to the organization of an international working group focused on harmonizing all aspects of the technique. During this workshop, a technical consensus was reached on the methods for cell permeabilization and immunophenotyping procedures. An assay was then designed that allowed comparison of two clones of anti‐ZAP‐70 antibody and the identification of the expression of this molecule in B, T, and NK cells identified in a four multicolor analysis. This procedure was applied to three stabilized blood samples, provided by the UK NEQAS group to all participating members of this study, in order to minimize variability caused by sample storage and shipment. Analysis was performed in 20 laboratories providing interpretable data from 14 centers. Various gating strategies were used and the ZAP‐70 levels were expressed as percentage positive (POS) relative to isotype control or normal B‐cells or normal T‐cells; in addition the levels were reported as a ratio of expression in CLL cells relative to T‐cells. The reported level of ZAP‐70 expression varied greatly depending on the antibody and the method used to express the results. The CLL/T‐cell ZAP‐70 expression ratio showed a much lower interlaboratory variation than other reporting strategies and is recommended for multicenter studies. Stabilization results in decreased expression of CD19 making gating more difficult and therefore stabilized samples are not optimal for multicentric analysis of ZAP‐70 expression. We assessed the variation of ZAP‐70 expression levels in fresh cells according to storage time, which demonstrated that ZAP‐70 is labile but sufficiently stable to allow comparison using fresh samples distributed between labs in Europe. These studies have demonstrated progress toward a consensus reporting procedure, and further work is underway to harmonize the preparation and analysis procedures.

Reconstitution of PTEN activity by CK2 inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect of human stromal cells in chronic lymphocytic leukemia

Evidence suggests that tumor microenvironment is critically involved in supporting survival of chronic lymphocytic leukemia (CLL) cells. However, the molecular mechanisms of this effect and the clinical significance are not fully understood. We applied a microenvironment model to explore the interaction between CLL cells and stromal cells and to elucidate the role of phosphatidylinositol 3 kinase (PI3-K)/Akt/phosphatase and tensin homolog detected on chromosome 10 (PTEN) cascade in this process and its in vivo relevance. Primary human stromal cells from bone marrow, lymph nodes, and spleen significantly inhibited spontaneous apoptosis of CLL cells. Pan-PI3-K inhibitors (LY294002, wortmannin, PI-103), isotype-specific inhibitors of p110α, p110β, p110γ, and small interfering RNA against PI3-K and Akt1 counteracted the antiapoptotic effect of the stromal cells. Induction of apoptosis was associated with a decrease in phosphatidylinositol-3,4,5-triphosphate, PI3-K-p85, and dephosphorylation of phosphatidylinositol-dependent kinase-1 (PDK-1), Akt1, and PTEN. Freshly isolated peripheral blood mononuclear cells from patients with CLL (n = 44) showed significantly higher levels of phosphorylated Akt1, PDK-1, PTEN, and CK2 than healthy persons (n = 8). CK2 inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole, apigenin, and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazol) decreased phosphorylation of PTEN and Akt, induced apoptosis in CLL cells, and enhanced the response to fludarabine. In conclusion, bone marrow microenvironment modulates the PI3-K/Akt/PTEN cascade and prevents apoptosis of CLL cells. Combined inhibition of PI3-K/Akt and recovery of PTEN activity may represent a novel therapeutic concept for CLL.

PDGFR blockade is a rational and effective therapy for NPM-ALK-driven lymphomas

Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkins lymphoma found in children and young adults. ALCLs frequently carry a chromosomal translocation that results in expression of the oncoprotein nucleophosmin–anaplastic lymphoma kinase (NPM-ALK). The key molecular downstream events required for NPM-ALK–triggered lymphoma growth have been only partly unveiled. Here we show that the activator protein 1 family members JUN and JUNB promote lymphoma development and tumor dissemination through transcriptional regulation of platelet-derived growth factor receptor-β (PDGFRB) in a mouse model of NPM-ALK–triggered lymphomagenesis. Therapeutic inhibition of PDGFRB markedly prolonged survival of NPM-ALK transgenic mice and increased the efficacy of an ALK-specific inhibitor in transplanted NPM-ALK tumors. Notably, inhibition of PDGFRA and PDGFRB in a patient with refractory late-stage NPM-ALK+ ALCL resulted in rapid, complete and sustained remission. Together, our data identify PDGFRB as a previously unknown JUN and JUNB target that could be a highly effective therapy for ALCL.

Increased levels of transforming growth factor-β1 in essential hypertension

BACKGROUND Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that has been linked to vascular remodeling processes, myocardial hypertrophy, and renal fibrosis. Recently a correlation between serum levels of TGF-beta1 and blood pressure (BP) levels in patients with end-stage renal disease was shown. In addition, it is not clear whether TGF-beta1 is a causative factor in the pathogenesis of essential hypertension and associated with hypertensive target organ damage (TOD). METHODS Using a TGF-beta1-specific sandwich ELISA, we compared plasma levels of active and total TGF-beta1 of 30 normotensive persons and 85 patients with essential hypertension with and without TOD, as measured by microalbuminuria or left ventricular hypertrophy. RESULTS Active and total TGF-beta1 levels were significantly higher in plasma of patients with essential hypertension than in normotensive controls (P < .05 and P < .01, respectively). However, neither active nor total TGF-beta1 correlated with systolic or diastolic BP (R2 < 0.14 for all parameters). Levels of active and total TGF-beta1 were significantly higher in hypertensive patients with than without TOD (P < .05). CONCLUSIONS Active and latent TGF-beta1 levels are markedly increased in plasma of hypertensive patients. We assume that TGF-beta1 contributes substantially to the development of TOD in essential hypertension, independent of BP levels.

Induction of apoptosis by proteasome inhibitors in B-CLL cells is associated with downregulation of CD23 and inactivation of Notch2.

Recently, proteasome inhibitors (PI) have attracted interest as novel anticancer agents in B-cell chronic lymphocytic leukemia (B-CLL). A prominent feature of B-CLL cells is the high expression of CD23, which is closely related to cell survival and is regulated by Notch2. Since several components of the Notch signaling cascade are tightly regulated by proteasomal degradation, we studied the effect of PI on Notch2 activity and CD23 expression. Exposure of B-CLL cells to PI led to induction of apoptosis, a time- and dose-dependent downregulation of CD23 expression and a decline in DNA binding of transcriptionally active Notch2. In contrast, the transcription factor NF-AT and its putative target gene CD5, which is highly expressed in B-CLL cells, were unaffected. When the late phase of PI-induced apoptosis was arrested by inhibition of caspase 3, the reduction of Notch2 activity was still observed, indicating that reduction of active Notch2 took place already during an earlier phase of apoptosis. Enforced expression of constitutively active Notch2 decreased PI-mediated apoptosis in a human B-cell line. These data indicate that downregulation of CD23 and loss of Notch2 activity are early steps in PI-induced apoptosis of B-CLL lymphocytes and may be part of the full apoptotic response.

TGF-β1 induces bone marrow reticulin fibrosis in hairy cell leukemia

The mechanisms that lead to reticulin fibrosis of bone marrow (BM) in hairy cell leukemia (HCL) are not fully understood. We therefore investigated the involvement of TGF-β1, a potent fibrogenic cytokine, in this process. Immunoassays revealed that TGF-β1 is present at higher concentrations in BM, serum, and plasma of HCL patients in comparison with healthy donors (P < 0.001). RT-PCR and immunofluorescence studies showed that TGF-β1 is overexpressed at the mRNA and protein levels in peripheral blood, spleen, and BM mononuclear cells and that hairy cells (HCs) are the main source of TGF-β1. Active TGF-β1 correlated significantly with grades of BM fibrosis, infiltration with HCs, and serum procollagen type III aminoterminal propeptide (PIIINP). Ex vivo studies demonstrated that TGF-β1 significantly enhances the production and deposition of reticulin and collagen fibers by BM fibroblasts. In addition, BM plasma of HCL patients increased the synthesis of type I and type III procollagens, the main components of reticulin fibers, at the mRNA and protein levels. This fibrogenic activity of BM plasma was abolished by neutralizing anti–TGF-β1 antibodies. These results show, for the first time to our knowledge, that TGF-β1 is highly expressed in HCs and is directly involved in the pathogenesis of BM reticulin fibrosis in HCL.

Deregulated expression of fat and muscle genes in B-cell chronic lymphocytic leukemia with high lipoprotein lipase expression.

Lipoprotein lipase (LPL) is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL) related to immunoglobulin VH gene (IgVH)mutational status. We determined gene expression profiles using Affymetrix U133A GeneChips in two groups of B-CLLs selected for either high (‘LPL+’, n=10) or low (‘LPL−’, n=10) LPL mRNA expression. Selected genes were verified by real-time PCR in an extended patient cohort (n=42). A total of 111 genes discriminated LPL+ from LPL− B-CLLs. Of these, the top three genes associated with time to first treatment were Septin10, DMD and Gravin (P⩽0.01). The relationship of LPL+ and LPL− B-CLL gene expression signatures to 52 tissues was statistically analyzed. The LPL+ B-CLL expression signature, represented by 64 genes was significantly related to fat, muscle and PB dendritic cells (P<0.001). Exploration of microarray data to define functional alterations related to the biology of LPL+ CLL identified two functional modules, fatty acid degradation and MTA3 signaling, as being altered with higher statistical significance. Our data show that LPL+ B-CLL cells have not only acquired gene expression changes in fat and muscle-associated genes but also in functional pathways related to fatty acid degradation and signaling which may ultimately influence CLL biology and clinical outcome.

Liver X receptors interfere with cytokine-induced proliferation and cell survival in normal and leukemic lymphocytes

Liver X receptors (LXRs) are nuclear receptors regulating lipid and cholesterol metabolism. Recent data indicate an additional role of LXR in immunity by controlling dendritic cell and T‐cell function and in breast and prostate cancer cells. Here, we show that LXR activation interferes with IL‐2 and IL‐7‐induced proliferation and cell cycle progression of human T‐cell blasts mainly through inhibited phosphorylation of the retinoblastoma protein and decreased expression of the cell cycle protein cyclin B. Comparable results were obtained with IL‐2‐dependent chronic lymphoblastic leukemia (CLL) T cells. Furthermore, we show for B‐CLL cells that LXR are functionally active and inhibit expression of survival genes bcl‐2 and MMP‐9, and significantly reduce cell viability, suggesting an interference of LXR with cytokine‐dependent CLL cell survival. In conclusion, our data reveal LXR as a potent modulator of cytokine‐dependent proliferation and survival of normal and malignant T and B lymphocytes. This novel LXR action could find clinical application in immunosuppressive and antileukemic therapies.