The Opposing Role of Histone Deacetylase 10 (HDAC10) and HDAC11 in Proliferation/Survival of Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1363-1363 ◽  
Author(s):  
Eva Sahakian ◽  
Bijal D. Shah ◽  
John Powers ◽  
Susan Deng ◽  
Oscar Merino ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Cell Line ◽  
Cell Biology ◽  
Lymphocytic Leukemia ◽  
B Cell Malignancies ◽  
Aberrant Expression ◽  
Over Expression

Abstract Abstract 1363 The role of HDACs in cell biology, initially limited to their effects upon histones, encompasses now more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution and the stage of cellular differentiation. Not surprisingly, HDACs have been shown to play important roles in normal B-cell biology and, aberrant expression of these proteins has been found in some B-cell malignancies1. However, the role of specific HDACs in regulation of pro-survival and cell-cycling genes in MCL and CLL still remains poorly understood. We therefore evaluated by RT-PCR the mRNA expression of specific HDACs in MCL and CLL cell lines and in primary cells from patients with these B-cell malignancies. Our analysis revealed a unique and opposing expression of HDAC10 and HDAC11 in these malignant B-cells. While HDAC11 over-expression was frequently found in MCL and CLL cells, in particular in patients with aggressive disease, an almost complete abrogation of HDAC10 was observed in malignant B-cells as compared to normal B-cell controls. These findings led us to explore the biological consequences of manipulating HDAC11 and HDAC10 in MCL and CLL cells. First, knocking-down HDAC11 (HDAC11KD) using lentiviral shRNA resulted in downregulation of cyclin D1, Cdkn1a (p21) and bcl-2. Furthermore, HDAC11KD MCL or CLL cells displayed a slower cell proliferation relative to non-target shRNA control cells. Cell cycle analysis revealed that HDAC11KD clones are arrested in G1. Conversely, over-expression of HDAC11 in the MCL cell line Z138c or in the CLL cell line MEC1 resulted in enhanced cell survival and increased proliferative capacity. Mechanistically, we have recently found that HDAC11 over-expression is associated with increased phosphorylation of STAT3, a known survival pathway in malignant B-cells. Second, HDAC10 belongs to the class II HDAC family and its biological functions remain largely unknown. Similar to our results in aggressive MCL and CLL, a decreased HDAC10 expression has been reported in patients with aggressive solid tumors2, suggesting that loss of HDAC10 expression might confer a survival advantage to malignant cells. Indeed, over-expression of HDAC10 in Z138c and MEC1 cells resulted in a rapid induction of cell death in vitro with only 5% of cells being alive at 48 hours. Our results highlight the need for a better understanding of the expression/function of specific HDACs in MCL and CLL biology. The findings of opposing roles for HDAC11 and HDAC10 in influencing cell survival and proliferation might explain the limited efficacy of pan-HDAC inhibitors (with their indiscriminate inhibition of multiple HDACs) in these B-cell malignancies, and provide support for the development of isotype-selective inhibitors targeting HDAC11. Disclosures: Chen-Kiang: Pfizer, Inc.: Research Funding.

UCH-L1 Provides An Essential Survival Signal in Malignant B-Cells by Enhancing NF-κB Activation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3338-3338
Author(s):  
Sajjad Hussain ◽  
Paul J. Galardy
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Myeloma Cell ◽  
Luciferase Reporter ◽  
Myeloma Cell Line ◽  
B Cell Malignancies ◽  
Over Expression ◽  
Lymphoid Malignancies ◽  
Fold Reduction

Abstract Using an activity-based approach, we previously described the over-expression of the deubiquitinase ubiquitin carboxy terminal hydroxylase 1 (UCH-L1) in several B-cell malignancies including multiple myeloma, Burkitt lymphoma, non-Hodgkin lymphoma and chronic lymphocytic leukemia. UCH-L1 over expression also correlates with a poor prognosis in non-lymphoid tumors including non-small cell lung, esophageal, colorectal and pancreatic cancers. Whether UCH-L1 expression contributes to the malignant phenotype in these conditions, however, has not been established. We therefore sought to determine the role of UCH-L1 in lymphoid malignancies through the use of lentivirusencoded short hairpin RNA (shRNA) targeting UCH-L1. When compared with a control non-silencing shRNA, reduced UCH-L1 levels are associated with a profound block in proliferation and loss of viability (>90%) in the UCH-L1 expressing myeloma cell line KMS11. The introduction of these shRNAs into the UCH-L1 negative myeloma cell line KMS12 does not affect proliferation or viability. The association of cell death with reduced UCH-L1 levels was further reinforced using a doxycycline inducible shRNA construct. KMS11 and KMS12 cells grow equally well following transduction and selection of stable viral integrants. However, upon induction of shRNA production with doxycycline, greater than 90% of KMS11 cells die within 48-hours timed with the depletion of UCH-L1 while KMS12 cells continue to proliferate. These results strongly suggest that UCH-L1 is essential for survival in UCH-L1 expressing myeloma cells. Given the critical role of NF-kB signaling in many lymphoid malignancies, we examined the impact of UCH-L1 depletion on NF-kB activity. Using a secreted luciferase reporter assay, we determined NF-kB activity in control and UCH-L1 depleted KMS11 cells. We observed a 5-fold reduction of NF-kB activity in UCH-L1 knockdown cells compared to control. In order to establish a correlation between loss of UCH-L1 expression and NF-kB down-regulation, cells transduced with an inducible shRNA were treated with doxycycline and monitored daily for UCH-L1 and NF-kB activation. We observed a direct relationship between the level of UCH-L1 and NF-kB activity in these cells with a 5-fold reduction in NF-kB activity by 48-hours accompanied by reduced viability. These effects are entirely reversible with the removal of doxycycline from the growth medium. Doxycycline induction had no effect on the NF-kB activity of the UCH-L1 negative cell line KMS12. Knockdown of UCH-L1 does not affect TNF-alpha-induced NF-kB activity in these cells suggesting that UCH-L1 modulates the non-canonical NF-kB pathway. A relationship between UCH-L1 and NF-kB is further suggested by observing a 3.8-fold increase in baseline NF-kB activity in HeLa cells over-expressing UCH-L1. These data strongly indicate that UCH-L1 enhances NF-kB through the non-canonical pathway. In order to determine whether UCH-L1 over-expression can by itself promote tumorigenesis in vivo, we have engineered a UCH-L1 transgenic mouse. These mice will provide us the opportunity to study the effect of UCH-L1 expression on spontaneous as well as induced (carcinogen or oncogene) tumorigenesis. Further studies are examining the effect of forced UCH-L1 expression on B-cell development. This study will provide a better understanding of the role of UCH-L1 in hematological malignancies and may validate UCH-L1 as an important drug target for B-cell malignancies.

scholarly journals Methylome-based cell-of-origin modeling (Methyl-COOM) identifies aberrant expression of immune regulatory molecules in CLL

2020 ◽  
Author(s):  
Justyna A. Wierzbinska ◽  
Reka Toth ◽  
Naveed Ishaque ◽  
Karsten Rippe ◽  
Jan-Philipp Mallm ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Biology ◽  
Disease Onset ◽  
Lymphocytic Leukemia ◽  
B Cell Differentiation ◽  
Cell Of Origin ◽  
Aberrant Expression ◽  
Aberrant Transcript ◽  
Epigenetic Patterns

ABSTRACTBackgroundIn cancer, normal epigenetic patterns are disturbed and contribute to gene expression changes, disease onset and progression. The cancer epigenome is composed of the epigenetic patterns present in the tumor-initiating cell at the time of transformation, and the tumor-specific epigenetic alterations that are acquired during tumor initiation and progression. The precise dissection of these two components of the tumor epigenome will facilitate a better understanding of the biological mechanisms underlying malignant transformation. Chronic lymphocytic leukemia (CLL) originates from differentiating B cells, which undergo extensive epigenetic programming. This poses the challenge to precisely determine the epigenomic ground-state of the cell-of-origin in order to identify CLL-specific epigenetic aberrations.MethodsWe developed a linear regression model, methylome-based cell-of-origin modeling (Methyl-COOM), to map the cell-of-origin for individual CLL patients based on the continuum of epigenomic changes during normal B cell differentiation.ResultsMethyl-COOM accurately maps the cell-of-origin of CLL and identifies CLL-specific aberrant DNA methylation events that are not confounded by physiologic epigenetic B cell programming. Furthermore, Methyl-COOM unmasks abnormal action of transcription factors, altered super-enhancer activities, and aberrant transcript expression in CLL. Among the aberrantly regulated transcripts were many genes that have previously been implicated in T cell biology. Flow cytometry analysis of these markers confirmed their aberrant expression on malignant B cells at the protein level.ConclusionsMethyl-COOM analysis of CLL identified disease-specific aberrant gene regulation. The aberrantly expressed genes identified in this study might play a role in immune-evasion in CLL and might serve as novel targets for immunotherapy approaches. In summary, we propose a novel framework for in silico modeling of reference DNA methylomes and for the identification of cancer-specific epigenetic changes, a concept that can be broadly applied to other human malignancies.

scholarly journals Association of CXCR4 with IgM and IgD BCR Isotypes: Role in B Cell Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1852-1852
Author(s):  
Eva Gentner ◽  
Andrea Nicola Mazzarello ◽  
Martin Becker ◽  
Antonella Nicolò ◽  
Valerio Renna ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cytoplasmic Tail ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
In Vitro Assays ◽  
B Cell Malignancies ◽  
Cxcr4 Signaling

Abstract B cell malignancies including chronic lymphocytic leukemia (CLL) and diffuse large B cell lymphoma (DLBCL) are age-associated diseases driven by clonal B cell proliferation. Signaling through B cell antigen receptor (BCRs) is dysregulated in these diseases. In addition to BCRs, chemokine receptors, such as CXCR4 and CXCR5, are used to predict clinical course. The chemokine receptor CXCR4 is expressed at different developmental stages of B cells, serving different homeostatic functions including migration. We previously reported that the cross-talk of CXCR4 and the BCR isotype IgD is supporting survival and activation of mature B cells in mice. In this process, the B cell marker and co-activator CD19 plays a pivotal role. Nevertheless, interaction of BCR with CXCR4 has not been analyzed in detail in B cell malignancies. In this study, we further elucidated the CXCR4 signaling in mouse as well as human B cell subsets including immature and mature B cells. Consistent with murine B cells, CXCR4 signaling in human B cells from healthy donors remains tightly linked to surface IgD-BCR expression, although CXCR4 is highly expressed in human IgG positive memory B cell compartment. Furthermore, proximity of CXCR4 and IgD in human mature B cells is reminiscent of that of mouse B cells. In contrast, IgD:CXCR4 proximity is skewed towards IgM:CXCR4 in CLL cells. In in vitro assays, unmutated (U)-CLL cells migrate better compared to mutated (M)-CLL. Nevertheless, our analyses reveal a frequent association of IgM:CXCR4 in M-CLL. Taking together our murine and human data, we propose that IgD:CXCR4 association is crucial for CXCR4 signaling in both CLL and healthy B cells. Apart from CXCR4, mutations within the immunoreceptor tyrosine-based activation motif (ITAM) residues of CD79a and CD79b are frequently associated with B cell malignancies including DLBCL. Knowing the potential role of BCR and its isotypes in CXCR4 induced signaling, we further analyzed the role of CD79a and CD79b. Here, we took advantage of transgenic mice, whose CD79a and CD79b cytoplasmic tails carrying ITAM motifs can be inducibly deleted. Our analysis reveals the indispensable role of the CD79b cytoplasmic tail, whose loss of function causes complete impairment of CXCR4 induced signaling in murine B cells. In contrast, loss of CD79a cytoplasmic tail partially blocks CXCR4 induced signaling, which could be rescued by CD19 co-stimulation. Extending our murine results, we established an in vitro read-out system to test the role of ITAM mutants derived from DLBCLs, as well as DLBCL-derived isotypes for analyzing their impact on CXCR4 signaling. Taking our findings together, IgD:CXCR4 association is crucial for CXCR4 signaling in CLL and healthy B cells. An increased association of IgM:CXCR4 in M-CLL compared to U-CLL suggests the necessity of IgD:CXCR4 for functional CXCR4 signaling. Furthermore, CD79b is crucial for CXCR4 induced signaling in mature B cells and loss of CD79b function abrogates CXCR4 signaling in mature B cells. Disclosures Chiorazzi: AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

scholarly journals Pharmacologic Inhibition with Duvelisib (IPI-145) and Genetic Inhibition of PI3K p110δ Antagonizes Intrinsic and Extrinsic Survival Signals in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3324-3324 ◽  
Author(s):  
Shuai Dong ◽  
Daphne Guinn ◽  
Jason A Dubovsky ◽  
Yiming Zhong ◽  
Amy Lehman ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Nk Cells ◽  
Cell Line ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Pi3k Signaling

Abstract Background: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen, and bone marrow. CLL cells display up-regulated B-cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton’s tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. Both the PI3K p110δ inhibitor, idelalisib, and the BTK inhibitor, ibrutinib, have received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies. Methods/Results: Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, duvelisib (IPI-145), which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of duvelisib in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM duvelisib for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to duvelisib, displaying about 20% decrease in viability at concentrations greater than 0.5μM; however, the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM duvelisib for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, duvelisib is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in the leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose-dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, duvelisib. Disclosures Dubovsky: Principia Inc.: Research Funding. Kutok:Infinity Pharmaceuticals, Inc.: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: a mechanism for survival

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2973-2979 ◽  
Author(s):  
Anne J. Novak ◽  
Richard J. Bram ◽  
Neil E. Kay ◽  
Diane F. Jelinek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocyte ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Aberrant Expression ◽  
B Lymphocyte Stimulator

B-cell chronic lymphocytic leukemia (B-CLL) is defined by the accumulation of CD5+ B cells in the periphery and bone marrow. This disease is not characterized by highly proliferative cells but rather by the presence of leukemic cells with significant resistance to apoptosis and, therefore, prolonged survival. B-lymphocyte stimulator (BLyS) is a newly identified tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis and it shares significant homology with another TNF superfamily member, APRIL. The striking effects of BLyS on normal B-cell maintenance and survival raises the possibility that it may be involved in pathogenesis and maintenance of hematologic malignancies, including B-CLL. In this study, we investigated the status of APRIL and BLyS expression, as well as their receptors, in this disease. All B-CLL patient cells studied expressed one or more of 3 known receptors for BLyS; however, the pattern of expression was variable. In addition, we demonstrate for the first time that B-CLL cells from a subset of patients aberrantly express BLyS and APRIL mRNA, whereas these molecules were not detectable in normal B cells. Furthermore, we provide in vitro evidence that BLyS protects B-CLL cells from apoptosis and enhances cell survival. Because these molecules are key regulators of B-cell homeostasis and tumor progression, leukemic cell autocrine expression of BLyS and APRIL may be playing an important role in the pathogenesis of this disease.

scholarly journals Expression and release of CD27 in human B-cell malignancies

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3430-3436 ◽  
Author(s):  
MH van Oers ◽  
ST Pals ◽  
LM Evers ◽  
CE van der Schoot ◽  
G Koopman ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Growth Factor Receptor ◽  
Lymphocytic Leukemia ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Grade ◽  
B Cell Malignancies ◽  
Variable Intensity ◽  
Hodgkin's Lymphomas

Abstract CD27, a transmembrane disulfide-linked 55-kD homodimer, belongs to the nerve growth factor-receptor family, a group of homologous molecules involved in lymphocyte differentiation and selection. It is expressed on mature thymocytes, peripheral blood T cells, and a subpopulation of B cells. We investigated the expression of CD27 on malignant B cells representative for a broad range of stages in physiologic antigen- independent and -dependent B-cell development. In normal lymphoid tissue CD27+ B cells were only found in the peripheral blood (29.8% +/- 10.8%, n = 13) and in germinal centers. With the exception of pro-B and the majority of pre-pre-B acute lymphocytic leukemias and of myelomas, CD27 expression of variable intensity was detected on almost all immature and mature malignant B cells tested. Moreover, using a sandwich enzyme-linked immunosorbent assay we could show the presence of sometimes very high (up to 6,000 U/mL; normal values < 190 U/mL) amounts of the soluble 28- to 32-kD form of CD27 (sCD27) in the sera of patients with B-cell malignancies. The highest levels of sCD27 were observed in patients with chronic lymphocytic leukemia and low-grade non-Hodgkin's lymphomas. Most importantly, both in transversal and longitudinal studies, we found a strong correlation between sCD27 levels in the serum and tumor load, indicating that sCD27 can be used as a disease-marker in patients with acute and chronic B-cell malignancies.

The Akt/Mcl-1 Pathway Plays a Prominent Role in Mediating Pro-Survival Signals Derived from the B-Cell Receptor in Chronic Lymphocytic Leukemia B-Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 341-341
Author(s):  
Pablo G. Longo ◽  
Luca Laurenti ◽  
Stefania Gobessi ◽  
Simona Sica ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Igm Antibodies ◽  
B Cell Survival ◽  
Sustained Activation ◽  
Constitutively Active

Abstract Studies of the immunoglobulin variable region gene repertoire have provided compelling evidence that antigen-stimulation through the B-cell receptor (BCR) plays a crucial role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). In addition, previous studies from our lab have shown that CLL B-cells become more resistant to spontaneous and chemotherapy-induced apoptosis following sustained engagement of the BCR with immobilized anti-IgM antibodies, which mimic stimulation with membrane-bound antigens. Investigation of downstream signaling pathways revealed that sustained BCR engagement induces prolonged activation of the PI3K/Akt and MEK/ERK pathways, which are key regulators of survival and proliferation in various cell types. To further define the role of sustained activation of the Akt and ERK kinases in regulating CLL growth and survival, we transfected constitutively active mutants of Akt (myr.Akt) and MEK2 in primary leukemic cells and evaluated changes in the expression of relevant apoptosis- and cell-cycle regulatory proteins. Introduction of constitutively active MEK2 resulted in activation of ERK, but did not induce significant changes in the levels of most investigated proteins (Bcl-2, Bcl-xL, Bim, Bax or Mcl-1). The only exception was the inhibitor of apoptosis protein XIAP, which showed increased expression in most but not all experiments. In contrast, transfection of myr.Akt showed a consistent increase in the levels of the antiapoptotic protein Mcl-1, which ranged from 1.5 to more than 4-fold higher levels with respect to cells transfected with control vectors. Increased expression of Mcl-1 was observed in all experiments and paralleled the rise in Mcl-1 that occurred following stimulation of CLL B-cells with immobilized anti-IgM antibodies. The increase in Mcl-1 protein levels was entirely due to post-transcriptional mechanisms, since quantification by real-time PCR did not show an increase in Mcl-1 mRNA levels. Constitutively active Akt also upregulated Bcl-xL and XIAP, although this increase was lower than the increase in Mcl-1. In addition, CLL cells transfected with myr.Akt showed induction of cyclin D3 and an increase in cell size and viability, indicating that sustained activation of Akt is required for both leukemic cell survival and cell cycle progression. To determine the relative importance of Mcl-1, Bcl-xL and XIAP in CLL B-cell survival, we downregulated expression of these proteins in primary CLL B-cells by RNA interference. Surprisingly, downregulation of Bcl-xL and XIAP had no effect on CLL B-cell survival. In contrast, silencing of Mcl-1 induced rapid and potent apoptosis in all investigated cases and abrogated the prosurvival effect of stimulation with immobilized anti-IgM antibodies. Together, these data provide direct evidence that pro-survival BCR signaling in CLL B-cells is mediated, at least in part, through the Akt/Mcl-1 pathway. In addition, they suggest that Mcl-1 could be an attractive candidate for targeting, either with small molecule inhibitors or with pharmacological agents that interfere with BCR signals propagated by the Akt kinase.

Chronic Lymphocytic Leukemia (CLL) Cells Require Microenvironmental Stimuli to Resist Apoptosis through Activation of STAT3 Mediated by VEGF.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1069-1069
Author(s):  
Iris Gehrke ◽  
Julian Paesler ◽  
Rajesh Kumar Gandhirajan ◽  
Regina Razavi ◽  
Alexandra Filipovich ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Lymphocytic Leukemia ◽  
Vegf Receptor ◽  
Mrna Levels ◽  
Activated State ◽  
Survival Effect

Abstract B-cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, but incompetent B-cells due to a decrease of apoptosis rather than an increase in proliferation. Vascular endothelial growth factor (VEGF) has been suggested to play an important role in this so called apoptotic block. However, so far little is understood whether VEGF is acting mainly as a microenvironmental stimulus and/or whether CLL cells themselves contribute to the enhanced apoptotic resistance by maintaining an autocrine VEGF loop. Moreover, it is unknown by which mechanisms VEGF prevents apoptosis and whether this can be circumvented by inhibition of VEGF signaling. By quantitative real time PCR we found no significant difference in mRNA VEGF levels in B-cells from CLL patients and healthy donors after isolation from blood. In contrast, ELISA revealed clearly increased levels of secreted VEGF in plasma of CLL patients and in the supernatant under culture conditions compared to healthy individuals. In addition, we found the VEGF receptor 2 (VEGFR2), which is existent in CLL and healthy B-cells, in a phosphorylated, hence activated state, to a significantly higher extent in CLL cells as assessed by intracellular phospho flow cytometry. In conclusion, despite its expression in healthy B-cells VEGF does not seem to be secreted and therefore, no VEGF receptor phosphorylation takes place. Whereas CLL cells exhibit a long life span in vivo, they die rapidly in vitro, suggesting major survival factors being existent in the CLL cells microenvironment. We found levels of secreted VEGF in supernatant decreasing with time in culture, going along with decreasing levels of phosphorylated VEGFR2 and increasing cell death as assessed by Annexin V-FITC/PI staining. This further supports the role of VEGF in CLL cell survival. Coculturing primary CLL cells with the bone marrow stromal derived cell line HS5 dramatically increased VEGF transcription and secretion and improved cell survival. Hence, VEGF expression in CLL cells is not only mediated by autocrine, but also paracrine stimuli involving bone marrow stromal. Knocking down VEGF in HS5 cells and subsequent coculture with CLL cells might prove the major role of VEGF in this survival supporting coculture setting. Besides coculturing also supplement of culture medium with recombinant human VEGF (rhVEGF) increased survival, but to a lesser extent than coculture, indicating a direct cell-cell interaction as advantageous. Furthermore, we found a downregulation of anti apoptotic proteins, such as X-linked inhibitor of apoptosis protein (XIAP), myeloid cell leukemia 1 (MCL1) and BclXL upon VEGF stimulation. Also cyclinD1 was upregulated as seen by immunoblotting. We further tried to discover the underlying mechanism of how VEGF mediates its pro survival effect and found STAT3 to become phosphorylated on tyrosine 705 upon VEGF stimulation. In CLL STAT3 is known to be constitutively phosphorylated on serine 727. This phosphorylation is not sufficient to induce target gene expression though. We could show that Y705 phosphorylation of STAT3 is responsible for upregulation of anti apoptotic BCLXL and cyclinD1. A PCR array detecting mRNA levels of 84 transcription factors in untreated and VEGF stimulated CLL cells shall provide more information about mechanistical details how VEGF mediates it pro survival effect. Since VEGF seems to be a major player in CLL cell survival it might be a suitable target to overcome the apoptotic block. In first experiments we found an induction of apoptosis after neutralization of VEGF or inhibition of the VEGF receptor. This additionally highlights the severe importance of VEGF in the apoptotic block in CLL cells. Therefore, VEGF might serve as an excellent therapeutic target in CLL.

Different Expression of FcgammaRIIb in Chronic Lymphocytic Leukemia and Human Normal B Lymphocytes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3134-3134
Author(s):  
Carol Moreno ◽  
Rajendra Damle ◽  
Sonia Jansa ◽  
Gerardo Ferrer ◽  
Pau Abrisqueta ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Surface Membrane ◽  
Memory B Cells ◽  
Lymphocytic Leukemia ◽  
Cd38 Expression ◽  
B Cell Subsets

Abstract The Fcgamma receptors (FcγRs) are a family of molecules that modulate immune responses. FcγRIIb is an inhibitory FcγR that bears immunoreceptor tyrosine-based inhibitory motifs which transduce inhibitory signals on coligation with the surface membrane Ig of the B-cell antigen receptor (BCR). The role of FcγRIIb in controlling B cell activation through inhibition of BCR signaling has been extensively studied in animal models. Nevertheless, data on FcγRIIb are scant in human normal and neoplastic B cells, this being due to the lack of a specific antibody for human FcγRIIb. Consequently, there is little information on this receptor in chronic lymphocytic leukemia (CLL). Considering the activated nature of CLL cells and the central role of the BCR in the biology of the disease, studies of FcγRs are warranted. We used a novel specific mAb directly conjugated with Alexa 488 fluorophore that solely reacts with the human FcγRIIb (MacroGenics, Inc.) to investigate the receptors expression on CLL and normal human B cells. The study population included 84 patients with CLL and 24 age- and sex-matched controls. FcγRIIb expression was assessed as the mean fluorescence intensity (MFI) of surface membrane staining. In CLL cells, FcγRIIb was measured on CD19+CD5+ cells in combination with CD38, CD49d or CD69. Normal B cells were immunostained for CD19, CD5, IgD and CD38 expression and B cell subsets: naïve (IgD+CD38−), activated (IgD+CD38+) and memory B cells (IgD−CD38−) were studied for their relative expression of FcγRIIb. FcγRIIb expression was found significantly higher in naïve B cells compared to activated and memory B cells [median MFI: 17420 (11960–21180) vs. 11.140 (7899–16970) and 11.830 (6984–17100); p&lt;0.001]. Significant differences were also observed between CD5− and CD5+ normal B cells. In contrast, FcγRIIb expression was lower in CLL cells than in CD5+ and CD5− normal B lymphocytes [median MFI: 6901(1034–42600), 10180 (5856–14820) and 12120 (7776–16040); p&lt;0.05)]. Interestingly, FcγRIIb expression was variable within individual CLL clones, this being higher in CD38+ and CD49d+ cells than in CD38− and CD49d− cells (p&lt;0.05). Furthermore, the highest density of FcγRIIb was observed on those cells which coexpressed CD38 and CD49d. In contrast, no significant differences were observed between FcγRIIb and the expression of the activation antigen CD69. Although CD69 and CD38 expression was significantly higher on unmutated IGHV cases, no correlation was found between FcγRIIb levels and IGHV mutational status. Similarly, there was no correlation between FcγRIIb and other poor prognostic variables such as ZAP-70 (≥20%), CD38 (≥ 30%) or high risk cytogenetics. Nevertheless, cases with ≥ 30% CD49d+ cells had higher FcγRIIb expression than those with &lt;30% CD49d+ cells (p=0.006). The findings presented in this study suggest a hierarchy of FcγRIIb expression in normal B-cells, CLL cells and their subpopulations: circulating normal CD5− B cells &gt; circulating normal CD5+ B cells &gt; circulating CD5+ CLL B cells. In addition, although FcγRIIb is present on all normal B cell subsets its expression is higher in naïve B cells. Furthermore, in CLL FcγRIIb density is greater in CD38+ and CD49d+ cells within the clone. Although CD49d and FcγRIIb on CLL clones is linked in a direct manner, there is no relationship with FcγRIIb density and IGHV mutations, ZAP-70, CD38 and unfavorable cytogenetic markers. Finally, the relationship between FcγRIIb expression on CLL cells and functional responses to BCR and other receptor-mediated signals deserve further investigation.

The Target Genes and Prognostic Significance of Mir-150 in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3859-3859
Author(s):  
Marek Mraz ◽  
Laura Z. Rassenti ◽  
Emanuela M. Ghia ◽  
Liguang Chen ◽  
Jessie-Farah Fecteau ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Biology ◽  
Fold Change ◽  
Lymphocytic Leukemia ◽  
Inverse Association ◽  
Expression Of Genes ◽  
Bcr Signaling ◽  
Indolent Disease

Abstract Abstract 3859 Chronic lymphocytic leukemia (CLL) is the first disease in which miRNAs (hsa-miR-15a-16–1) were directly linked to cancer pathogenesis (Calin et al. PNAS, 2002). We and others have also shown that expression of certain miRNAs associates with disease activity in patients with CLL (Calin et al. NEJM, 2005; Mraz et al. Blood, 2012; Mraz et al. Leukemia, 2009). Moreover, patients with more aggressive disease have CLL cells that generally express unmutated IGHV and/or ZAP-70 and have a miRNA expression profile that differs from that of CLL cells from patients with indolent disease (Calin et al. NEJM, 2005). However, we still have very limited understanding of how miRNAs affect CLL cell-biology and expression of genes that play a critical role in either promoting or arresting the disease. We used pooled samples from 10 CLL patients to screen (TaqMan miRNA Cards-ABI, 750 miRNAs) for abundantly expressed miRNAs that could hypothetically influence CLL B cell biology. We identified miR-150 as the most abundant miRNA in CLL cells and also as being strongly expressed when compared to CD19+ blood lymphocytes of normal adults (N=5, P=0.008). This miRNA already has been reported to influence the differentiation and gene expression of normal B cells (Xiao et al. Cell, 2007) suggesting its possible relevance for CLL B cell biology. We examined additional CLL cell samples (N=168) and confirmed high miR-150 levels and also noted heterogeneity in its expression between CLL cells of patients with aggressive versus indolent disease. In our cohort, CLL cells of patients that expressed ZAP-70 (20% cut-off, N=74) or had unmutated IGHV (N=72) expressed significantly lower median-levels of miR-150 (fold change −1.7 and −2.0 respectively, p<0.005). Moreover, the lower levels of miR-150 also directly associated with higher response to stimulation of B-cell receptor (BCR) on CLL cells with anti-IgM (P<0.05, N=36, quantified by flow cytometric measurement of calcium mobilization). To understand the gene network regulated by miR-150 in CLL we performed array-based transcriptome analyses (HG-U133 Plus 2.0, Affymetrix) of 110 patient samples, which identified differential expression of 215 genes between CLL cells expressing low versus high levels of miR-150 (SAM analysis of upper and lower terciles). Thirty-eight of these 215 genes (17%) are predicted targets of miR-150 (determined by TargetScan, www.targetscan.org). Two well annotated genes (GAB1 and FOXP1) have evolutionary conserved binding sides for miR-150 in their 3‘UTRs, suggesting the possible importance of miR-150 in their regulation. GAB1 is an adaptor molecule and plays a key role in variety of cell signaling pathways (PLCγ, Ras/Erk, PI3K/Akt, CrkL). Interestingly, GAB1 modulates PI3K/Akt-pathway through binding domain identical to Bruton’s tyrosine kinase (Rameh et al. JBC, 1997) and is a key molecule involved in regulating BCR-signaling (Ingham et al. JBC, 1998, 2001), a process that factors prominently in the pathogenesis and progression of CLL. FOXP1 is an essential participant in the transcriptional regulatory network of B lymphopoiesis and has been identified as playing a role in disease progression of other B-cell lymphomas (Hu et al. Nat Immunol, 2006). The immunoblot analysis of GAB1 and FOXP1 in CLL cells confirmed their higher protein levels in cases with low miR-150 expression (P<0.005, fold change >10.0). Importantly, cells with higher expression of GAB1 or FOXP1 were more responsive to BCR stimulation in vitro (P<0.01, N=36) and higher expression of each associates with shorter overall survival (OS) (13.9 vs. 22.7 years, 13.9 vs. 21.1 years; N=168; P<0.05). Most notably, a reverse trend was observed for miR-150, where higher levels (>median) were associated with significantly longer OS (not-reached vs. 13.9 years, N=168, P=0.006). Additionally, the expression level of miR-150 was an independent predictor of OS and time to first treatment (TTFT) in multivariate analyses, which included IGHV status, ZAP-70, CD38, Rai stage, gender, and age (OS HR: 3.4 [CI 1.4–8.6], P=0.009; TTFT HR: 2.3 [CI 1.3–4.2], P=0.004). We conclude that there is an inverse association between high-risk disease and expression of miR-150, which may reflect its capacity to regulate the expression of genes encoding proteins that may contribute to BCR-signaling and/or survival of CLL B cells. Disclosures: No relevant conflicts of interest to declare.

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