The Hsp90 Inhibitor 17-DMAG Increases SOCS3 and Regulates Cytokine Production, Migration and Cell Death in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1362-1362
Author(s):  
Erin K Hertlein ◽  
Timothy L. Chen ◽  
Rainer Claus ◽  
Christoph Plass ◽  
Amy Lehman ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Gene Silencing ◽  
Cell Survival ◽  
Cytokine Production ◽  
Transcriptional Silencing ◽  
Hsp90 Inhibitor ◽  
Lymphocytic Leukemia ◽  
Cytokine Signaling ◽  
Cxcr4 Signaling

Abstract Abstract 1362 Epigenetic or transcriptional silencing of important tumor suppressors has been described to contribute to cell survival and tumorigenesis in chronic lymphocytic leukemia (CLL). We investigated gene silencing in CLL using gene expression microarray analysis, and found that over 2000 genes are repressed more than 2-fold in CLL B cells compared to normal B cells, including genes involved in cell signaling and survival (Jun/Fos signaling, ATF family, cyclin dependent kinases and suppressors of cytokine signaling). In particular, the suppressor of cytokine signaling protein 3 (SOCS3) was decreased 60-fold in CLL B cells compared to peripheral blood B cells from normal donors. Despite this profound effect, few therapeutic approaches have focused on reversing this gene silencing in CLL. NF-κB has been shown to transcriptionally silence gene expression in several types of cancer, and our group has previously identified a similar role for this transcription factor in CLL. In addition, we have previously shown that the Hsp90 inhibitor 17-DMAG targets NF-κB signaling to induce apoptosis in CLL through transcriptional down-regulation of oncogenes such as MCL1 and BCL2. However, our microarray analysis revealed that treatment with 17-DMAG also leads to the re-expression of nearly 200 genes silenced in CLL compared to normal B cells. One of the genes significantly silenced in CLL and re-expressed by 17-DMAG is SOCS3. This increase in SOCS3 was evident as early as 8 hours following treatment with 17-DMAG, and peaking at 16–24 hours just prior to 17-DMAG induced cell death (up-regulated 5.6 fold at 8 hours, 59.8 fold at 16 hours, and 25.7 fold at 24 hours). The magnitude of induction in CLL cells was significantly greater than SOCS3 induction in peripheral blood B cells from normal donors, which correlates with a decreased apoptotic response of normal B cells to 17-DMAG (25.7 fold up-regulated in CLL versus 3.5 fold in normal B cells). While little is known about its regulation or functional impact in CLL, SOCS3 has been shown to be methylated in solid tumors as well as myeloid leukemia, leading to aberrant cytokine production and cell survival. While it is known that promoter hypermethylation and subsequent gene silencing contributes to CLL disease progression, we found that there was no significant methylation of the SOCS3 promoter in CLL compared to normal B lymphocytes, indicating an alternative mechanism of SOCS3 silencing in CLL. In order to further characterize the downstream effect of SOCS3 regulation, we investigated the pathways known to be regulated by this protein, specifically IL-6 and CXCR4 signaling. We found that 17-DMAG prevents phosphorylation of STAT3 induced by IL-6 stimulation, which leads to decreased production of pro-survival cytokines including negative feedback by decreasing IL-6 itself. While STAT3 is a known Hsp90 client protein, the effect on phosphorylation of STAT3 was evident before a decrease in the total protein was observed, indicating a distinct effect on the signaling pathway independent of Hsp90's role as a protein chaperone. SOCS3 has also been shown to prevent phosphorylation of focal adhesion kinase (FAK) and therefore block both integrin and CXCR4 signaling pathways. We found that 17-DMAG prevents constitutive phosphorylation of FAK in primary CLL cells, and subsequently reduces AKT phosphorylation following recombinant SDF-1 stimulation. In order to determine if 17-DMAG inhibits migration of CLL cells towards recombinant SDF-1 as well as the direct signaling through the CXCR4 receptor, we performed transwell migration assays and found that 17-DMAG significantly inhibits migration towards both recombinant SDF-1 and CXCL13 (migration towards CXCL12, 12.4% with Vehicle vs. 8.9% with 17-DMAG, p=0.0061, towards CXCL13, 12.4% with Vehicle vs. 6.1% with 17-DMAG, p<0.0001). Similar results were obtained by over-expression of SOCS3 in a CLL B cell line, suggesting that 17-DMAG inhibits migration through an increase in SOCS3. Based on these results, we suggest that 17-DMAG reverses gene silencing in CLL, and through re-expression SOCS3 inhibits the migration and signaling associated with SDF-1/CXCR4, an important factor in the tumor microenvironment that contributes to CLL cell survival. Therefore Hsp90 inhibitors represent a novel approach to target transcriptional silencing in CLL and other B cell lymphoproliferative disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization of TET and IDH gene expression in chronic lymphocytic leukemia: comparison with normal B cells and prognostic significance

2016 ◽  
Vol 8 (1) ◽  
Author(s):  
Michaël Van Damme ◽  
Emerence Crompot ◽  
Nathalie Meuleman ◽  
Marie Maerevoet ◽  
Philippe Mineur ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Prognostic Significance ◽  
Lymphocytic Leukemia

Human Antagonistic Anti-CD40 Antibody, CHIR-12.12, Inhibits CD40-Mediated Survival of Chronic Lymphocytic Leukemia B Cells Leading to Cellular Apoptosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2965-2965 ◽  
Author(s):  
Anu Cherukuri ◽  
Edward Kadel ◽  
Sang H. Lee ◽  
Cheryl Goldbeck ◽  
Carla Heise ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Signaling Pathways ◽  
P38 Mapk ◽  
Hodgkin’S Lymphoma ◽  
Caspase 3 ◽  
Lymphocytic Leukemia ◽  
Hodgkin's Lymphoma ◽  
Non Hodgkin's Lymphoma

Abstract CD40 and CD40 ligand (CD40L) interaction is a key regulator of B-chronic lymphocytic leukemia (CLL) survival. CD40 activation leads to binding with tumor necrosis factor receptor-associated factors (TRAFs) and the subsequent activation of multiple downstream signaling pathways involved in cellular proliferation and survival. We have generated a novel fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, using XenoMouse® mice (Abgenix, Inc). CHIR-12.12 blocks CD40L binding to CD40 and inhibits CD40L-induced proliferation/survival of normal human B cells, primary CLL cells, and primary non-Hodgkin’s lymphoma (NHL) cells. We have also demonstrated that it has highly potent antibody-dependent cellular cytotoxicity (ADCC) against primary CLL and non-Hodgkin’s lymphoma cells. We have now investigated its effects on primary CLL cell survival. Soluble human CD40L prolongs primary CLL cell survival in culture, and treatment with CHIR-12.12 inhibits this survival when measured 48–72 hours after addition of CHIR-12.12. CD40L-mediated survival is associated with activation and phosphorylation of Akt, p38 MAPK, ERK, and IkB kinases a and b. Additionally, the anti-apoptotic proteins Mcl-1, Bcl-xl, and XIAP are induced, and markers of apoptosis (cleaved PARP and Caspase-3) are reduced. In contrast, CHIR-12.12 treatment of CD40L-stimulated primary CLL cells ex vivo inhibited downstream phosphorylation of Akt, p38 MAPK, ERK, and IkB kinases (IKK) a and b. Additionally, CHIR-12.12 treatment resulted in induction of cleaved caspase-3 and PARP, and reduction of XIAP, Mcl-1, and Bcl-xl expression, ultimately leading to CLL cell apoptosis. These results demonstrate that CHIR-12.12 inhibits CD40L-mediated signaling pathways and cell survival and could be a potential therapeutic treatment for CLL. CHIR-12.12 is currently in a Phase I clinical study for CLL.

Introduction of Constitutivelly Active Akt in Primary CLL B-Cells Results in Upregulation of Mcl-1, Bcl-XL and Cyclin D3 and Promotes Leukemic Cell Survival.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2805-2805
Author(s):  
Pablo Longo ◽  
Stefania Gobessi ◽  
Luca Laurenti ◽  
Simona Sica ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Survival ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia ◽  
Cyclin D3 ◽  
Leukemic Cells ◽  
Interleukin 4 ◽  
Antiapoptotic Proteins ◽  
Sustained Activation ◽  
Constitutively Active

Abstract The PI3K/Akt and Raf/MEK/ERK pathways are key regulators of various cellular responses, including proliferation, survival, differentiation, migration and malignant transformation. These pathways are activated in chronic lymphocytic leukemia B-cells by a number of survival or growth stimulatory signals, such as immobilized anti-IgM antibodies, interleukin-4, phorbol-ester, CXCL12, or stimulatory CpG oligonucleotides. Moreover, enhanced activation of Akt has been implicated in the pathogenesis of the CLL-like disorder that develops in mice transgenic for the TCL1 oncogene. To further delineate the relative contribution of the PI3K/Akt and Raf/MEK/ERK pathways in regulating leukemic cell growth and survival, we introduced constitutively active Akt or constitutively active MEK2 in primary CLL B-cells by nucleofection. Expression of constitutively active Akt consistently promoted survival, as evidenced by a higher percentage of Annexin V/PI negative cells after 48 hours in culture (median 52%) compared to samples transfected with control vector (median 31%). Immunoblot analysis of several important antiapoptotic proteins revealed that enforced activation of Akt upregulates Mcl-1 and Bcl-xL, whereas no changes were observed in the levels of Bcl-2. Expression of constitutively active Akt also induced an increase in size and granularity of the leukemic cells, indicating increased metabolic activity. These changes were associated with significant induction of cyclin D3, indicating that activation of Akt is required for both leukemic cell survival and cell cycle progression. In contrast, introduction of constitutively active MEK2 induced sustained activation of ERK, but showed only a modest increase in the percentage of viable CLL B-cells (median 36%) and no significant changes in the levels of any of the investigated antiapoptotic proteins. These experiments provide direct evidence that sustained activation of Akt promotes leukemic cell survival and upregulates Mcl-1, an antiapoptotic protein that has been associated with resistance to chemotherapy in patients with CLL.

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.

Reduced c-FOS Is Associated with Poor Prognosis and Clinical Course in Chronic Lymphocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2433-2433
Author(s):  
Christopher C Oakes ◽  
Rainer Claus ◽  
Christopher Schmidt ◽  
Yoon Jung Park ◽  
Michael Boutros ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Gene Family ◽  
Conflicts Of Interest ◽  
Mapk Pathway ◽  
Substantial Reduction ◽  
Lymphocytic Leukemia ◽  
Prognostic Indicators ◽  
Autologous Serum ◽  
Isolated Cells

Abstract Abstract 2433 We have previously shown that silencing of the tumor suppressor gene, DAPK1, by genetic and epigenetic mechanisms is associated with the pathogenesis of CLL. To elucidate genes and pathways involved in DAPK1 transcriptional regulation, genome-wide siRNA screens were performed using a novel transgene reporter system involving a stably-integrated BAC construct expressing luciferase under the control of the DAPK1 locus. c-FOS and other members of the FOS gene family were identified as positive regulators, implicating the AP-1 pathway in DAPK1 transcription. A subsequent comprehensive examination of the FOS, JUN and ATF gene families in CLL and healthy B cells was performed. As the expression of the AP-1 gene family is highly subject to fluctuations in stress and mitogenic stimuli, freshly isolated cells were cultured using autologous serum conditions, allowing for a uniform establishment of baseline gene expression. This work reveals an alteration of the relative composition of AP-1 transcription factors in CLL, demarked by a substantial reduction in c-FOS gene expression. We find that the inducibility of c-FOS in CLL following MAPK activation by TPA (ERK-MAPK) is impaired 7.0-fold relative to healthy B cells and is completely abrogated following anisomycin (p38-MAPK) stimulation. The level of c-FOS induction following TPA activation can be used to clearly segregate CLL patients into two non-overlapping groups, those with low (mean=4.0-fold reduced expression; range=3.0–7.2; n=9) and very low (mean=21.7-fold reduced expression; range=17.6–36.7; n=8) following one hour induction versus healthy B cells. The very low c-FOS induction cases are characterized by poor prognostic indicators (IGHV unmutated (0/9 in low vs. 5/8 in very low); 17p, 6q deletion (0/9 in low vs. 3/8 in very low); and a substantially shorter time to progression (102.7±40.4 months in low vs. 10.8±8.6 in very low). Patients with very low c-FOS induction also demonstrate elevated c-MYC induction following activation. TPA-dependent ERK phosphorylation and activation of other immediate early genes, such as EGR1 and c-JUN, is intact in both CLL groups, absolving MAPK pathway dysfunction as a relevant c-FOS silencing mechanism. Reduced c-FOS expression can be partially explained, in the majority of very low c-FOS inducible cases, by elevated levels of miR-155 and miR-221 targeting the c-FOS transcript. Reduced expression of c-FOS and the resulting reconfiguration of AP-1 transcription factor composition may be involved in the pathogenesis of CLL and the subsequent silencing of DAPK1. Disclosures: No relevant conflicts of interest to declare.

Sensitivity to Wnt Pathway Inhibition in CLL Is Associated with Specific Gene Expression Signatures

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 801-801
Author(s):  
Lili Wang ◽  
Alex K Shalek ◽  
Jellert Gaublomme ◽  
Nir Yosef ◽  
Jennifer R Brown ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Cell Viability ◽  
B Cell ◽  
Cell Survival ◽  
Wnt Pathway ◽  
Differentially Expressed ◽  
Differential Sensitivity ◽  
Specific Gene ◽  
Disease Heterogeneity

Abstract Abstract 801 We have recently found that the Wnt/b-catenin signaling pathway plays a key role in chronic lymphocytic leukemia (CLL). We were, however, intrigued by the question of whether this aberrant pathway may function differently in independent leukemias, and contribute to disease heterogeneity. To assess differential activity of the Wnt pathway across patients, we tested the effects of blocking Wnt activation on CLL cell survival. We knocked down a key downstream gene, LEF1, which is the most differentially expressed gene in CLL compared to normal B cells (based on gene expression microarrays). Addressing this question requires genetic manipulation of primary normal and malignant human B cells, and yet these cells are notoriously difficult to transfect. We therefore focused on developing a method for introducing siRNAs into normal and malignant B cells. We adapted a novel delivery system consisting of vertical silicon nanowires (SiNWs, Shalek et al PNAS 2010) that penetrate the plasma membrane in a minimally invasive fashion and deliver biomolecular cargo directly into the cytoplasm. We achieved consistent and reliable delivery of fluorescently labeled siRNAs (at 50–200 pmol) into normal and CLL B cells. siRNA was delivered to >90% of cells with >85% cell viability remaining after 48 hours. We used this platform to knockdown LEF1 in 20 CLL-B and 5 normal CD19+ B cell samples, and examined cell survival 48 hours after siRNA delivery using an ATP-based CellTiter-Glo assay. Indeed, our studies revealed a heterogeneous response among CLL-B cells to LEF1 inhibition. As a group, CLL-B cells were significantly more sensitive to LEF1 knockdown with a survival rate of 77% (12% s.e.m) compared to 97% (13% s.e.m) in normal B cells. CLL B cells from different patients showed differential sensitivity to LEF1 knockdown, with 8 non-responders, 8 intermediate responders and 4 strong responders (i.e. significant death). Sensitivity to LEF1 inhibition did not correlate with known CLL cytogenetic prognostic factors. To determine if the differential response to LEF1 knockdown was associated with specific gene signatures, we examined gene expression data generated from CLL-B cells from 12 (4 strong, 3 intermediate, and 5 non-responders) of the 20 CLLs tested (using the Affymetrix U133 Plus 2 Array). To increase statistical power, we used each CLL's expression profile (using only genes that showed variability across samples) to create clusters of ∼19 CLLs that showed similar expression profiles (using microarray data from our compendium of 177 additional CLLs). We further reduced the number of genes to ∼4000 genes by retaining only those whose expression levels were significantly different in at least one associated cluster relative to normal CD19+ B cell controls (T-test, FDR<10−4; p-values converted using the Benjamini-Hochberg method). These analyses led to the identification of several hundred genes whose expression correlated significantly with LEF1 knockdown's effect on cell viability. Analysis of these differentially expressed genes identified several potentially important pathways. Ongoing analyses include the identification and validation of a molecular signature for this effect. This signature could enable rapid identification of patients who would be most responsive to therapy with LEF1 inhibitors, which are under development along with other Wnt pathway inhibitors. Disclosures: No relevant conflicts of interest to declare.

Impact of Lenalidomide on Gene Expression Profiles of Malignant and Immune Cells in Patients with Chronic Lymphocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 976-976 ◽  
Author(s):  
John C. Riches ◽  
Ajanthah Sangaralingam ◽  
Shahryar Kiaii ◽  
Tracy Chaplin ◽  
Demet Cekdemir ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Family Member ◽  
Cell Function ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Lymphocytic Leukemia ◽  
Healthy Donors

Abstract Abstract 976 Lenalidomide has recently been demonstrated to have significant activity in chronic lymphocytic leukemia (CLL). Its mechanism of action in this disease is not well understood, but it is thought to act primarily by enhancing anti-tumor immunity and reducing production of pro-tumoral factors in the CLL microenvironment. We have previously demonstrated alterations in the expression of cytoskeletal genes in T-cells from patients with CLL and have subsequently shown that these changes translate into a deficit in T-cell function, due to impaired actin polymerization resulting in defective immunological synapse formation. Treatment of both autologous T-cells and CLL cells with lenalidomide was necessary to repair this defect, suggesting that this may be a key component of this agent's activity in CLL. Therefore we examined the effect of lenalidomide on the global gene expression profiles of isolated B-cells and T-cell subsets from CLL patients and healthy donors. Peripheral blood mononuclear cells from patients with untreated CLL or healthy donors were cultured in the presence of 1 μM lenalidomide or vehicle control for 48 hours. The lymphocyte subsets were isolated, followed by RNA extraction and gene expression profiling using the Affymetrix HGU133Plus2.0 platform. Lenalidomide treatment had similar effects on gene expression in T-cells from both patients with CLL and healthy donors. The most prominent changes in expression were of genes involved in cytoskeletal signaling including a 20-fold increase in WASF1 (Wiskott Aldrich Syndrome protein family, member 1), and greater than 2-fold increases in the expression of Rac-family member RHOC, (Ras homolog gene family, member C), actin binding proteins CORO1B (Coronin 1B), PARVA (Parvin alpha), and the Rho guanine nucleotide exchange factors (GEFs), ARHGEF5 and ARHGEF7. We also observed changes in genes regulating integrin signaling including PXN (Paxilin) and FAK (Focal adhesion kinase), and a shift towards Th1 differentiation with upregulation of TNF, IL-12R, and IL-18R. In addition, we noted increased expression of the transcription factors IKZF1, IKZF4 and IRF4, genes involved in the Ikaros pathways that are essential for hematopoiesis and control of lymphoid proliferation. These changes in gene expression provide further evidence that an important mechanism of action of lenalidomide is the upregulation of the actin cytoskeletal network including Rho-GTPases and integrin activation signaling, and are consistent with our previous observations concerning the functional repair of T-cells in CLL. Initial analysis of the effect of lenalidomide on the gene expression profiles of the CLL B-cells showed similar changes to those previously described in vivo from CLL patients receiving single agent lenalidomide in a clinical trial (Chen et al. JCO 2010). In our system, lenalidomide treatment resulted in a greater than 2-fold upregulation of 189 genes, and a greater than 2-fold downregulation of 85 genes in CLL B-cells. We observed increased expression of several genes belonging to the TNF superfamily including TNF-α, OX40L, and APRIL, and the receptors DR5, DCR2, and OX40. Many of these are known to mediate apoptosis signaling, and we also observed increased expression of pro-apoptotic genes such as FAS, BID (BH3 interacting domain death agonist), HRK (Harakiri), and CFLAR (CASP8 and FADD-like apoptosis regulator), and cell cycle regulators CDKN1A and CDKN1C (Cyclin-dependent kinase inhibitors 1A and 1C). Lenalidomide also upregulated expression of several genes of known importance in the CLL microenvironment, including the chemokines CCL3 and CCL4, CD40, CD274 (PD-L1), CD279 (PD-1), and adhesion molecules LFA3 and ICAM1. The effect of lenalidomide on the gene expression profiles of normal B-cells was less marked, with greater than 2-fold upregulation of 51 genes and downregulation of 12 genes. However, we did observe that lenalidomide treatment induced upregulation of genes involved in cytoskeletal pathways such as RND1 (Rho family GTPase 1), RHOQ (Ras homolog gene family, member Q), and MYO1B (myosin 1B). In conclusion, investigation of the effect of lenalidomide on gene expression profiling in CLL suggests that the drug acts both to enhance T-cell function, and to render the CLL cells more susceptible to immune cell mediated killing. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

FcμR Triggering Activates BCR Signaling Pathways and Increases Survival of CLL Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2015-2015
Author(s):  
Stefania Gobessi ◽  
Binu K Sasi ◽  
Gabriele Pozzato ◽  
Idanna Innocenti ◽  
Luca Laurenti ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Survival ◽  
Signaling Pathways ◽  
Opposite Effect ◽  
Lymphocytic Leukemia ◽  
Apoptosis Resistance ◽  
Membrane Expression ◽  
Bcr Signaling ◽  
Cells Cultured ◽  
Stimulation Of

Abstract Soluble IgM was recently shown to activate B cell receptor (BCR) signaling pathways in chronic lymphocytic leukemia (CLL) B cells and protect them from spontaneous and CpG DNA-induced apoptosis (Wagner M et al, Blood 2016, 127:436-48; Gobessi S et al, Blood 2015, 126:4131, abstract 4131). The exact mechanism behind these effects is still unknown, but interaction of the leukemic BCRs with internal epitopes in the framework regions of soluble IgM has been considered as a possible explanation. An alternative explanation for these effects is that they are mediated by binding of soluble IgM to the Fcμ receptor (FcμR), which is highly overexpressed in CLL compared to normal B cells (Li FJ et al, Blood. 2011; 118:4902-9) and was recently shown to physically interact with the BCR in normal murine B cells (Ouchida R et al, J Immunol. 2015; 194:3096-101). To evaluate the possibility that triggering of the FcμR is responsible for the activation of BCR signaling pathways and for the increased apoptosis resistance of CLL cells, we first investigated the viability of CLL cells cultured for 72 hours with or without soluble human IgM or Fcμ fragment. Both soluble human IgM and Fcμ significantly increased the viability of CLL cells with respect to unstimulated control, suggesting that triggering of the FcμR is sufficient to increase CLL cell survival (n=28, % viable IgM-stimulated: 52.6±18.3, % viable Fcμ-stimulated: 55.0±14.8, % viable unstimulated: 43.7±17.0, p<0.001 for IgM- and Fcμ-stimulated vs unstimulated, p=n.s. for IgM-stimulated vs Fcμ-stimulated). We next investigated whether triggering of FcμR can activate BCR signaling pathways. CLL cells were stimulated for 10 min with Fcμ or anti-IgM and the levels of phospho-SYK, phospho-AKT, phospho-ERK (n=7) and intracellular Ca2+ (n=5) were analyzed by immunoblotting and flow cytometry, respectively. Increased phosphorylation of SYK, AKT and ERK and increased Ca2+ flux were detected in 5 and 4 of the investigated samples, respectively. The effects of Fcμ stimulation appeared greater in samples that showed a weaker response to anti-IgM stimulation, suggesting that FcμR and BCR signaling are reciprocally regulated. Because IL-4 was recently shown to increase surface IgM expression on CLL cells (Aguilar-Hernandez MM et al, Blood. 2016, 127:3015-25; Guo B et al, Blood 2016, 128:553-562), we next investigated whether it will have an opposite effect on FcμR expression. Stimulation of CLL cells (n=7) for 48 hours with IL-4 resulted in a mean 2.4 fold reduction in surface FcμR expression and a 3.9 fold increase in surface IgM expression compared to unstimulated cells (P<0.001 and P=0.016, respectively). Since IL-4 is produced by T cells, which typically interact with CLL cells in lymph nodes (LN), we next compared surface FcμR and IgM expression in two paired LN and peripheral blood (PB) CLL samples. Interestingly, in both cases the levels of surface FcμR were lower on LN than PB CLL cells, whereas no difference was detected in the expression of surface IgM. We next compared signaling in CLL cells cultured in the presence or absence of IL-4. As previously reported, CLL cells cultured in the presence of IL-4 showed a considerably greater increase in anti-IgM induced phosphorylation of SYK, PLCγ2 and AKT compared to controls. Interestingly, greater activation of SYK, PLCγ2 and AKT was also observed when FcμR was downregulated by RNA interference in one primary CLL sample, suggesting that FcμR expression could be in part responsible for the reduced capacity of PB CLL cells to respond to anti-IgM stimulation. In summary, this study shows that stimulation of FcμR by soluble IgM increases CLL cell survival and activates BCR signaling pathways, presumably because the two receptors are physically associated on the cellular membrane. Expression of FcμR is higher on PB than LN CLL cells and is negatively regulated by IL-4, which has an opposite effect on surface IgM expression. Preliminary data suggest that FcμR expression may reduce the capacity of the BCR to respond to external ligand. The purpose of such a mechanism is currently unclear, but one possibility could be to prevent inappropriate activation of PB CLL cells by (auto)antigens in the absence of co-stimulatory signals. Disclosures Efremov: Gilead: Honoraria.

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