Heat shock protein-90 inhibitor, NVP-AUY922, is effective in combination with fludarabine against chronic lymphocytic leukemia cells cultured on CD40L-stromal layer and inhibits their activated/proliferative phenotype

2012 ◽  
Vol 53 (11) ◽  
pp. 2314-2320 ◽  
Author(s):  
O. Giles Best ◽  
Stephen P. Mulligan
Keyword(s):  
Heat Shock ◽  
Chronic Lymphocytic Leukemia ◽  
Heat Shock Protein ◽  
Heat Shock Protein 90 ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Stromal Layer ◽  
Cells Cultured

scholarly journals ZAP-70 is a novel conditional heat shock protein 90 (Hsp90) client: inhibition of Hsp90 leads to ZAP-70 degradation, apoptosis, and impaired signaling in chronic lymphocytic leukemia

Blood ◽  
2005 ◽  
Vol 106 (7) ◽  
pp. 2506-2512 ◽  
Author(s):  
Januario E. Castro ◽  
Carlos E. Prada ◽  
Olivier Loria ◽  
Adeela Kamal ◽  
Liguang Chen ◽  
...  
Keyword(s):  
Heat Shock ◽  
Chronic Lymphocytic Leukemia ◽  
Heat Shock Protein ◽  
Heat Shock Protein 90 ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Hsp90 Inhibitors ◽  
B Cell Receptor Signaling ◽  
High Binding Affinity

AbstractThe zeta-associated protein of 70 kDa (ZAP-70) is expressed in patients with aggressive chronic lymphocytic leukemia (CLL). We found that ZAP-70+ CLL cells expressed activated heat-shock protein 90 (Hsp90) with high binding affinity for Hsp90 inhibitors, such as 17-allyl-amino-demethoxy-geldanamycin (17-AAG), whereas normal lymphocytes or ZAP-70- CLL cells expressed nonactivated Hsp90. Activated Hsp90 bound and stabilized ZAP-70, which behaved like an Hsp90 client protein only in CLL cells. Treatment with Hsp90 inhibitors such as 17-AAG and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) induced ZAP-70 degradation and apoptosis in CLL cells but not in T cells, and also impaired B-cell receptor signaling in leukemia cells. Transduction of ZAP-70- CLL cells with an adenovirus encoding ZAP-70 activated Hsp90 and specifically rendered the leukemia cells sensitive to 17-AAG. These data indicate that Hsp90 is necessary for ZAP-70 expression and activity; that ZAP-70 is unique among Hsp90 clients, in that its chaperone-dependency is conditional on the cell type in which it is expressed; and also that ZAP-70 is required for cell survival and signaling in CLL. Additionally, ZAP-70 expression in CLL cells confers markedly heightened sensitivity to 17-AAG or 17-DMAG, suggesting that these or other Hsp90 inhibitors could be valuable therapeutically in patients with aggressive CLL. (Blood. 2005;106:2506-2512)

scholarly journals Analysis of heat shock protein 70 and in vitro apoptosis in B-chronic lymphocytic leukemia cells by flow cytometry

1998 ◽  
Vol 10 (2) ◽  
pp. 101-109
Author(s):  
Tsunayuki KAKIMOTO ◽  
Atsuko NAGASAWA ◽  
Taijiro ISHIYAMA ◽  
Kunihiko FUKUCHI ◽  
Nobuyoshi TSURUOKA
Keyword(s):  
Flow Cytometry ◽  
Heat Shock ◽  
Chronic Lymphocytic Leukemia ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells

Lenalidomide Abrogates the Protective Influence of Nurse-Like Cells on Primary Chronic Lymphocytic Leukemia Cells In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3116-3116 ◽  
Author(s):  
Danelle F. James ◽  
Maryann R. Betty ◽  
Ruzbeh Mosadeghi ◽  
Thomas J. Kipps
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
Cell Survival ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Response To Treatment ◽  
Leukemia Cells ◽  
High Dependency ◽  
Cells Cultured

Abstract Lenalidomide (3-(4-amino-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione)) is an agent approved for treatment of patients with del 5q myelodysplastic syndromes and previously treated multiple myeloma. Lenalidomide has been found in early clinical trials to have potential therapeutic activity in patients with relapsed chronic lymphocytic leukemia (CLL). The mechanism(s) whereby this drug is active in CLL is unknown. In particular, studies to date have not found lenalidomide to have any direct cytotoxic activity on CLL cells in vitro. This has stimulated speculation that this agent might adversely affect the positive influence of the microenvironment on leukemia-cell survival. We and others have observed that cells found in the leukemia microenvironment can support CLL-cell survival in vitro. One such type of cells are nurse-like cells (NLC), which can differentiate from the CD14-positive blood mononuclear cells of CLL patients into large, round adherent cells that can attract and support CLL cell survival in vitro for weeks, if not longer. We evaluated the effects of lenalidomide on primary leukemia-cell survival in vitro when the CLL cells from different patients (N=21) were cultured alone or together with NLC generated as previously described [Tsukada Blood 2002]. We assessed the in-vitro activity of lenalidomide on primary CLL cells from 21 patients, in duplicate in a series of 6 experiments. Lenalidomide at concentrations of 0.1μM-200μM did not significantly impact the survival of CLL cells that were cultured alone for up to 12 days. Analysis of cell surface markers revealed increased expression of CD38 at 36 hours in 5/5 lenalidomide treated CLL samples compared with untreated cells (MFIR 5.7 +/− .86 vs. 3.4 +/− .83 p=.003). We observed sustained upregualtion of CD40 and regulation of CXCR4 in the majority of cells treated with lenalidomide. When cultured with NLC, the survival of CLL cells was comparable to or significantly higher than that of CLL cells cultured alone 62.4% vs. 51% (+/−3% SEM n=21 p [<] 0.0005). The addition of lenalidomide at concentrations of 0.1μM and greater to co-cultures of NLC and CLL cells caused specific reductions in CLL cell survival to levels similar to or lower than that of CLL cells cultured without NLC. In the presence of NLC, lenalidomide at 1μM reduced CLL cell viability compared to control (41.5% vs. 56% +/−4% p [<] 0.0005 paired student t test n=13). For most patients the levels of CLL cell viability on days 4 through 8 in the co-cultures with lenalidomide was significantly lower than those of CLL cells co-cultured with NLC in the absence of lenalidomide. As such, this study reveals that physiologic concentrations of lenalidomide might abrogate the protective influence of NLC on CLL cell survival in vitro and potentially in vivo. Conceivably, those patients who have leukemia cells displaying a high dependency on NLC for survival in vitro also might be most likely to experience a favorable clinical response to treatment with lenalidomide. This hypothesis will be tested in a prospective manner with a planned clinical trial evaluating lenalidomide for treatment of CLL through the CLL Research Consortium.

Heat-Shock Protein Signature Is Associated with Refractory Chronic Lymphocytic Leukemia Cells From Different In Vivo Microenvironments,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3866-3866
Author(s):  
Payal Gupta ◽  
Amit K. Mittal ◽  
Dennis D Weisenburger ◽  
Philip Bierman ◽  
Shantaram S Joshi
Keyword(s):  
Bone Marrow ◽  
Heat Shock ◽  
Chronic Lymphocytic Leukemia ◽  
Heat Shock Protein ◽  
Peripheral Blood ◽  
Treatment Cycle ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Activation Markers ◽  
Protein Signature

Abstract Abstract 3866 Chronic Lymphocytic Leukemia (CLL) is a monoclonal B-cell disorder with accumulation of leukemic cells in peripheral blood, bone marrow and lymphoid organs. It presents with a heterogeneous clinical course. Many patients survive long periods of time without any need for treatment, whereas other patients show resistance to treatment or relapse soon after administration of therapy. Although some prognostic markers such as mutational status of immunoglobulin variable heavy chain, chromosomal abnormalities, CD38 levels, or ZAP-70 expression may help predict at initial diagnosis which patients will have more aggressive disease, the exact factors that can determine chances of remission in CLL are still not clear, making treatment challenging. Furthermore, CLL remains an incurable disease, necessitating a way for controlling its progression. Identifying novel molecular signatures associated with refractory CLL disease may help devise targeted treatment strategies and thus may prolong survival times and prevent the progression of CLL in relapsed patients. Considering this, we performed gene expression profiling (GEP) on peripheral blood (PB), bone marrow (BM) and lymph node (LN) samples collected at the time of diagnosis. We divided CLL samples into 3 groups based on their response to treatment; i) Stable CLL group: asymptomatic patients requiring no treatment, ii) Treated but stable CLL group: patients required treatment but had stable disease for at least one year after the end of the treatment cycle, and iii) Relapsed CLL: patients who relapsed within a year of end of the treatment cycle. Significance analysis of microarray (SAM) revealed that the heat-shock protein (HSP) signature (HSJ2, HSP70, HSP90, HSP60, HSP10, HSP 105, HSP40, HSP27, HSPA2, HSJ1, HSF4, HSPCA), BCR signaling pathway (JUN, NFATC4, NFKBIE, PPP3CB, TRAF3, CD81, CCT4), activation markers (CD81, CD83) and MMPs (MMP3, MMP9) were overexpressed in relapsed PB-CLL (n=3) compared to stable PB-CLL (n=6) and treated but stable PB-CLL (n=10). Overexpression of heat-shock protein signature genes were further observed in additional relapsed PB-CLL (n=6) group compared to other two PB-CLL (n=22) group. Interestingly, the HSP signature was consistently overexpressed in relapsed BM-CLL (n=6) and LN-CLL (n=12) compared to stable and treated but stable BM-CLL (n=11) and LN-CLL (n=3) groups. HSPs are considered chaperones of tumorigenesis and known to enhance survival, migration, and proliferation of tumor cells which may contribute to relapse in patients. Furthermore, the HSPs genes (HSP90 and HSP70) were significantly overexpressed in LN-CLL as compared to PB-CLL which implies important role of the microenviroment in rendering CLL refractory. To investigate the link between the expression of the individual genes with the aggressiveness of the disease, Kaplan-Meier log-rank tests were performed. We found that the higher expression of HSP90A, HSP90B, HSJ, and MMP9 were significantly (p<0.05) associated with shorter time to treatment. In summary, our study suggests that HSP genes are overexpressed in refractory CLL patients and thus are promising targets to improve clinical outcome. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differential heat shock protein localization in chronic lymphocytic leukemia

2009 ◽  
Vol 87 (3) ◽  
pp. 467-476 ◽  
Author(s):  
N. C. Dempsey ◽  
F. Leoni ◽  
H. E. Ireland ◽  
C. Hoyle ◽  
J. H. H. Williams
Keyword(s):  
Heat Shock ◽  
Chronic Lymphocytic Leukemia ◽  
Heat Shock Protein ◽  
Protein Localization ◽  
Lymphocytic Leukemia ◽  
Differential Heat

BMS-936564 (MDX1338): A Fully Human Anti-CXCR4 Antibody Induces Apoptosis in an in Vitro Model of Stromal – Leukemia Cell Interaction for Chronic Lymphocytic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2887-2887
Author(s):  
Manoj Kumar Kashyap ◽  
Deepak Kumar ◽  
Harrison Jones Jones ◽  
Michael Y. Choi ◽  
Johanna Melo-Cardenas ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Leukemia Cell ◽  
Cell Interaction ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Drug Induced ◽  
Induced Apoptosis ◽  
Cells Cultured

Abstract Abstract 2887 Chronic lymphocytic leukemia (CLL) remains incurable despite advances in the biology and treatment of this disease. Current data support the notion that resistance to therapy is promoted by a “protective” tumor microenvironment in which non-leukemia cells produce factors that enhance the resistance of CLL cells to spontaneous or drug-induced apoptosis. One such factor is the chemokine CXCL12, which interacts with its receptor CXCR4 on CLL cells to promote cancer cell survival. To examine the therapeutic potential of blocking CXCL12-CXCR4 interactions, we studied the effect of BMS-936564, a fully human IgG4 anti-CXCR4 antibody, using an in vitro co-culture model of human bone marrow derived stomal-NKter cells – leukemia cell interaction. Such stromal-NKter cells secrete CXCL12 and enhance the resistance of CLL cells to apoptosis in vitro. We observed that primary CLL cells co-cultured with stromal-NKter cells had significantly greater viability than CLL cells cultured alone (20–60% above baseline at 48 hours). Moreover, CLL cells co-cultured with stromal cells had enhanced resistance to drug-induced apoptosis. We found that BMS-936564 antibody at concentrations of 2–200nM could enhance the rate of apoptosis of CLL cells cultured alone or in the presence of stromal cells. CLL cells that expressed unmutated IgVH genes or ZAP-70 appeared equally susceptible to treatment with BMS-936564 as did CLL cells that lack these adverse prognostic markers, as did CLL cells that harbored deletions in 17p13.2 and that were resistant to chemotherapeutic agents, such a fludarabine monophosphate. BMS-936564 antibody inhibited CXCL12 mediated F-Actin polymerization in CLL cells at lower concentrations (20–200nM) compared to AMD-3100 (Mozobil), a small molecule CXCR4 inhibitor (50–150μM). In addition, AMD-3100 did not induce apoptosis in CLL cells (10–300μM). In summary, we observed that the anti-CXCR4 antibody BMS-936564 inhibited CXCL12 mediated activation of the CXCR4 receptor in CLL cells and induced apoptosis in leukemia cells. The pro-apoptotic activity of BMS-936564 was observed in cells cultured alone or together with stromal cells suggesting that this antibody had direct cytotoxic effect on leukemia cells and that it can overcome the protective tumor microenvironment. More over, the activity of BMS-936564 was independent of the presence of poor prognostic factors such as del(17p) suggesting that its mechanism of action is P53 independent. These findings show evidence that the CXCR4-CXCL12 pathway is a valid therapeutic target in CLL and provide additional biological rationale for ongoing clinical trials in CLL and other hematological malignancies using BMS-936564. Disclosures: Kuhne: Bristol-Myers Squibb: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Kipps:Abbott: Consultancy, Research Funding.

scholarly journals Heat shock protein 70 regulates Tcl1 expression in leukemia and lymphomas

Blood ◽  
2013 ◽  
Vol 121 (2) ◽  
pp. 351-359 ◽  
Author(s):  
Eugenio Gaudio ◽  
Francesco Paduano ◽  
Apollinaire Ngankeu ◽  
Francesca Lovat ◽  
Muller Fabbri ◽  
...  
Keyword(s):  
T Cell ◽  
Heat Shock ◽  
Chronic Lymphocytic Leukemia ◽  
Heat Shock Protein ◽  
B Cell ◽  
Ataxia Telangiectasia ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
Ataxia Telangiectasia Mutated ◽  
Prolymphocytic Leukemia

Abstract T-cell leukemia/lymphoma 1 (TCL1) is an oncogene overexpressed in T-cell prolymphocytic leukemia and in B-cell malignancies including B-cell chronic lymphocytic leukemia and lymphomas. To date, only a limited number of Tcl1-interacting proteins that regulate its oncogenic function have been identified. Prior studies used a proteomic approach to identify a novel interaction between Tcl1 with Ataxia Telangiectasia Mutated. The association of Tcl1 and Ataxia Telangiectasia Mutated leads to activation of the NF-κB pathway. Here, we demonstrate that Tcl1 also interacts with heat shock protein (Hsp) 70. The Tcl1-Hsp70 complex was validated by coimmunoprecipitation experiments. In addition, we report that Hsp70, a protein that plays a critical role in the folding and maturation of several oncogenic proteins, associates with Tcl1 protein and stabilizes its expression. The inhibition of the ATPase activity of Hsp70 results in ubiquitination and proteasome-dependent degradation of Tcl1. The inhibition of Hsp70 significantly reduced the growth of lymphoma xenografts in vivo and down-regulated the expression of Tcl1 protein. Our findings reveal a functional interaction between Tcl1 and Hsp70 and identify Tcl1 as a novel Hsp70 client protein. These findings suggest that inhibition of Hsp70 may represent an alternative effective therapy for chronic lymphocytic leukemia and lymphomas via its ability to inhibit the oncogenic functions of Tcl1.

Targeting the CXCR4-CXCL12 Pathway Using an Anti-CXCR4 IgG1 Antibody (PF-06747143) in Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4162-4162
Author(s):  
Manoj K. Kashyap ◽  
Carlos I. Amaya-Chanaga ◽  
Deepak Kumar ◽  
Michael Y. Choi ◽  
Laura Z. Rassenti ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Microenvironment ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Advisory Board ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Induce Cell Death ◽  
Igg1 Antibody ◽  
Cells Cultured

Abstract Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults in the Western world. This leukemia is not curable and resistance to therapy is promoted by factors present in the tumor microenvironment including the chemokine CXCL12 (SDF-1), which interacts with its receptor CXCR4 and is thought to promote cancer cell survival. Here we explored the therapeutic potential of blocking CXCL12-CXCR4 interactions using PF-06747143, a humanized IgG1 antibody specific for CXCR4, which is expressed at high levels by CLL cells. Using primary leukemia cells from CLL patients, we found that PF-06747143 inhibited CXCL12-induced cell migration and blocked cytoskeletal changes via F-actin polymerization similar to AMD-3100 (Mozobil, a small molecule inhibitor of CXCR4). In addition, PF-06747143 induced apoptosis on CLL cells cultured alone or in the presence of human bone marrow-derived stromal cells (stroma-NK-tert). The pro-apoptotic activity of PF-06747143 was independent of high-risk prognostic factors including IGHV mutation status, ZAP-70 expression or TP53 mutation / 17p-deletion. Interestingly, AMD-3100, which binds and inhibits signaling through CXCR4, did not induce cell death in CLL or any of the cell lines tested. PF-06747143 did not induce apoptosis on normal B and T cells, and the ability of this anti-CXCR4 antibody to induce cell death on CLL cells appeared to be dependent on the crosslinking of CXCR4. This was supported by the fact that a Fab only fragment derived from PF-06747143 did not induce apoptosis despite of its high binding affinity for CXCR4. We observed that PF-06747143 induced complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in CLL cells. However, this antibody did not induce caspase activation but rather its cell death activity appeared to be dependent on the production of reactive oxygen species (ROS) in leukemia cells. This effect was similar to that observed with other ROS dependent antibodies such as obinutuzumab (Gazyva). ROS induction was observed with PF-06747143, but not its Fab derived fragment and preceded apoptosis suggesting that this is critical component of its mechanism of action. We evaluated synergism of PF-06747143 with other CLL therapeutic agents and observed that this antibody synergized with fludarabine, bendamustine, ibrutinib and rituximab in the majority of CLL patient samples tested. In summary, our studies showed that PF-06747143, a CXCR4 IgG1 antibody is a potent inhibitor of the CXCR4-CXCL12 pathway and induces cell death primarily in CLL cells but not in normal lymphocytes. The cytotoxic effect of PF-06747143 was similar in CLL cells cultured alone or with stromal cells, suggesting that this antibody has the potential to overcome the protective effect of the tumor microenvironment. We also showed that PF-06747143 induced programmed cell death on CLL cells was dependent on ROS production and that this antibody synergized with agents currently used for the treatment of CLL patients. Overall, these findings highlight the biological relevance of the CXCR4-CXCL12 pathway in CLL, and provide rationale for clinical evaluation of PF-06747143 in CLL and other cancers. Disclosures Choi: Gilead: Consultancy, Other: Advisory Board, Speakers Bureau; AbbVie: Consultancy, Other: Advisory Board, Research Funding. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.

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