Homoharringtonine reduced Mcl-1 expression and induced apoptosis in chronic lymphocytic leukemia

Abstract Homoharringtonine (HHT) is a plant alkaloid that inhibits the elongation phase of translation that is currently in clinical trials. Because the intrinsically short-lived antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) has been reported to support the survival of chronic lymphocytic leukemia (CLL) cells, we hypothesized that inhibition of protein synthesis by HHT would decrease Mcl-1 expression and induce apoptosis in CLL. In primary CLL cells, HHT induced significant apoptosis independent of the prognostic characteristics of the patients. This was associated with inhibition of translation and decreased Mcl-1 levels in CLL cells. Mcl-1 reduction was evident as early as 2 hours and continued to decrease in the next 6-8 hours, whereas cell death started in 2 hours and continued to increase for 24 hours. Reduction of the Mcl-1 level was due to translation inhibition and proteasome degradation rather than to transcription inhibition or caspase cleavage. HHT and the transcription inhibitor SNS-032 induced synergistic cell killing. Although stromal cells induced Mcl-1 expression and protected CLL cells from the toxicity of fludarabine, this induction was reversed by HHT, which overcame stromal cell–mediated protection. Thus, these results provide a rationale for clinical development of HHT in CLL as single agent or in combinations.

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Sorafenib-Induced Apoptosis of Chronic Lymphocytic Leukemia Cells Is Associated with Downregulation of RAF and Myeloid Cell Leukemia Sequence 1 (Mcl-1)

Molecular Medicine ◽

10.2119/molmed.2011.00164 ◽

2011 ◽

Vol 18 (1) ◽

pp. 19-28 ◽

Cited By ~ 27

Author(s):

Jessie-F. Fecteau ◽

Ila S. Bharati ◽

Morgan O’Hayre ◽

Tracy M. Handel ◽

Thomas J. Kipps ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Myeloid Cell ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Cell Leukemia ◽

Induced Apoptosis

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Standardizing Co-Culture Conditions Between Chronic Lymphocytic Leukemia Cells and Marrow Stromal Cells: Towards a Reliable and Reproducible System to Assess Cell Adhesion-Mediated Drug Resistance

Blood ◽

10.1182/blood.v112.11.3149.3149 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3149-3149

Author(s):

Antonina Kurtova ◽

Maite P. Quiroga ◽

William G. Wierda ◽

Michael Keating ◽

Jan A. Burger

Keyword(s):

Bone Marrow ◽

Drug Resistance ◽

Cell Adhesion ◽

Chronic Lymphocytic Leukemia ◽

Stromal Cells ◽

Lymphocytic Leukemia ◽

Marrow Stromal Cells ◽

Hpv E6 ◽

Induced Apoptosis

Abstract Contact between chronic lymphocytic leukemia (CLL) cells and accessory stromal cells in tissue microenvironments is considered to play a major role in regulating CLL cell survival and disease progression. Stromal cells of various origins and species, and variable stromal-CLL cell ratios have been used in the past to study CLL-stromal cell interactions and to assess cell-adhesion mediated drug resistance (CAM-DR). Because of the heterogeneity of the currently used in vitro systems to study CLL-MSC interactions, and the importance of these co-culture systems for development and testing of novel agents, we tested a panel of murine and human MSC lines for their capacities to support CLL cell survival and CAM-DR, using various CLL-MSC ratios and fludarabine (F-ara-A) to induce CLL cell apoptosis. We tested four murine, non-transformed MSC lines derived from bone marrow: M210B4, KUM4, ST-2 and KUSA-H1. Also, we tested three human transformed cell lines: Stroma-NKtert, derived from bone marrow and immortalized by human telomerase reverse transcriptase (hTERT), UE6E7-T2 derived from bone marrow and transformed with human papilloma viruses (HPV) E6, E7 and hTERT, and UCB408E6E7Tert33 derived from umbilical cord blood and transformed with hTERT and HPV E6, E7. CLL cells were isolated from peripheral blood of untreated patients and each cell line was tested with at least three different patients according to the following protocol: viability of CLL was tested after 24, 48 and 72 hours by flow cytometry after staining with DiOC6 and propidium iodide. The following conditions were assayed on each of the MSC lines: CLL cells in suspension culture, CLL cells in suspension culture with 10 mM F-ara-A, CLL cells in co-culture with MSC, and CLL cells in co-culture with MSC and with 10 mM F-ara-A. Firstly, we performed titration experiments in order to identify the most appropriate ratio between stromal and CLL cells, using CLL-MSC ratios of 5:1, 10:1, 20:1, 50:1 and 100:1. We found a decline in MSC-derived CLL cell protection at the highest ratio of 100:1, suggesting that ratios of 50:1 or lower provide optimal conditions for in vitro assays. Results shown in Table 1 were assayed using a 20:1 ratio and represented relative viabilities when compared to untreated controls (mean±SEM). Regarding the protective effect of different MSC, we found that all MSC lines demonstrated remarkable protection of CLL cells from spontaneous and F-ara-A-induced apoptosis. We also found that stromal cells that had round shape morphology and easily formed confluent monolayer (M210B4, KUSA-H1, Stroma-NKTert) showed more prolonged protective effect in comparison to cell lines with more spindle shaped morphology (ST-2, KUM4, UE6E7-T2). The failure of UE6E7-T2 and UCB408E6E7Tert33 to demonstrate long-term protection of CLL cells could be related to their own sensitivity to F-ara-A. In this comparative study we demonstrated that both murine and human MSC provide substantial and comparable levels of protection from spontaneous and drug-induced apoptosis. CLL:MSC ratios of 50:1 or lower can be considered ideal for co-culture experiments. Further experiments have to be done to determine the levels of MSC-derived protection in a larger series of CLL samples and in different laboratories for validation. Collectively, in these co-culture assays we can study CLL-MSC interactions and CLL drugs under more standardized conditions that may allow us to evaluate the efficacy of new treatments that target the CLL microenvironment. Time points 24 hours 48 hours 72 hours +Flu + MSC + MSC +Flu +Flu + MSC + MSC +Flu +Flu +MSC + MSC +Flu M210B4 85.2±2.4 117.2±5.0 110.5±4.9 30.8±12.6 138.1±9.5 113.0±2.2 5.2±3.1 138.1±5.1 120.4±3.4 ST-2 93.6±3.0 99.9±2.6 103.1±0.5 51.6±9.4 111.9±2.6 89.8±8.7 13.9±6.3 112.6±5.7 87.0±16.4 KUM-4 93.6±3.0 106.4±1.8 104.2±1.9 51.6±9.4 112.4±2.6 100.8±2.8 13.9±6.3 111.8±6.7 88.5±11.4 KUSA-H1 79.4±7.4 125.1±3.7 118.2±2.0 33.9±10.9 136.0±3.6 107.2±7.0 11.3±6.1 133.6±5.4 84.9±7.6 Stroma-NKTert 79.3±7.0 118.6±7.0 111.0±7.0 30.5±9.5 130.7±9.5 115.6±8.0 7.1±4.3 133.0±11.5 122.7±9.0 UE6E7-T2 79.3±7.0 113.4±3.9 109.3±3.0 30.5±9.5 118.4±4.8 85.0±7.1 7.1±4.3 119.2±6.9 51.0±10.1 UCB408 E6E7Tert33 81.5±7.2 120.2±5.4 111.8±2.7 36.7±9.4 123.7±6.3 86.7±7.7 8.5±6.7 119.7±6.1 50.8±13.0 Table 1. Flu: fludarabine (10mM/ml), MSC: marrow stromal cells

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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 ◽

10.1182/blood.v120.21.2887.2887 ◽

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.

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Microenvironmental stromal cells abrogate NF-κB inhibitor-induced apoptosis in chronic lymphocytic leukemia

Haematologica ◽

10.3324/haematol.2017.165381 ◽

2017 ◽

Vol 103 (1) ◽

pp. 136-147 ◽

Cited By ~ 8

Author(s):

Carl Philipp Simon-Gabriel ◽

Katharina Foerster ◽

Shifa Saleem ◽

Dorothee Bleckmann ◽

Marco Benkisser-Petersen ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Stromal Cells ◽

Lymphocytic Leukemia ◽

Induced Apoptosis

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Valproic Acid Induces Apoptosis in Chronic Lymphocytic Leukemia Cells through Activation of the Death Receptor Pathway and Potentiates TRAIL Response.

Blood ◽

10.1182/blood.v108.11.4949.4949 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4949-4949

Author(s):

Laurence Lagneaux ◽

Nicolas Gillet ◽

Alain Delforge ◽

Marielle Dejeneffe ◽

Basile Stamatopoulos ◽

...

Keyword(s):

Valproic Acid ◽

Chronic Lymphocytic Leukemia ◽

Mononuclear Cells ◽

Single Agent ◽

Death Receptor ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Potent Inducer ◽

Mutational Status ◽

Induced Apoptosis

Abstract Background: The anti-leukemic in vitro activity of valproic acid (VPA), a commonly used antiepileptic agent, was tested on lymphocytes derived from 40 patients with chronic lymphocytic leukemia (CLL) (Binet stage A=34, B=3, C=3). These patients had not been previously treated or remained untreated for the previous 6 months. Combined analysis of ZAP-70, CD38 and IgVH mutational status was performed for each patient. Methods: Mononuclear cells were incubated with VPA at 1, 5 and 10 mM for 24 hours. Cell viability was assessed by trypan blue exclusion assay, apoptosis by annexin V/propidium iodide(PI) labelling and PI staining after cell permeabilisation. Caspase activation was studied by flow cytometry analysis after cell treatment with selective caspase inhibitors. Results: Exposure of CLL cells to VPA resulted in dose-dependent cytotoxicity and apoptosis in all CLL patients tested. VPA-treatment induced apoptotic changes in CLL cells including phosphatidylserine (PS) externalisation and DNA fragmentation. The mean apoptotic rate was similar between IgVH mutated and unmutated patients or ZAP-70+/ZAP-70- cases. VPA induced apoptosis by the extrinsic pathway involving engagement of the caspase-8 dependent cascade. Although CLL cells are commonly resistant to death receptor-induced apoptosis, VPA increased significantly the sensitivity of leukemic cells to TRAIL (tumor necrosis factor α-related apoptosis-inducing ligand). In addition, VPA overcomed the prosurvival effects of bone marrow stromal cells. Conclusions: These data indicate that VPA, at the pharmacological concentration of 1 mM, is a potent inducer of apoptosis in CLL and should be further explored as a single agent. Also the combination of VPA and TRAIL may be a promising approach in the treatment of CLL.

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Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells

Blood ◽

10.1182/blood-2004-12-4918 ◽

2005 ◽

Vol 106 (5) ◽

pp. 1824-1830 ◽

Cited By ~ 202

Author(s):

Meike Burger ◽

Tanja Hartmann ◽

Myriam Krome ◽

Justyna Rawluk ◽

Hirokazu Tamamura ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Stromal Cells ◽

Chemokine Receptors ◽

Actin Polymerization ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Mitogen Activated Protein Kinase ◽

Cxcr4 Antagonists ◽

Induced Apoptosis

Abstract Growth and survival of chronic lymphocytic leukemia (CLL) B cells are favored by interactions between CLL and nontumoral accessory cells. CLL cells express CXCR4 chemokine receptors that direct leukemia cell chemotaxis. Marrow stromal cells or nurselike cells constitutively secrete CXCL12, the ligand for CXCR4, thereby attracting and rescuing CLL B cells from apoptosis in a contact-dependent fashion. Therefore, the CXCR4-CXCL12 axis represents a potential therapeutic target in CLL. We evaluated the most active CXCR4-specific antagonists (T140, TC14012, TN14003) for their capacity to inhibit CXCL12 responses in CLL cells. T140, or its analogs, inhibited actin polymerization, chemotaxis, and migration of CLL cells beneath stromal cells. CXCL12-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) was abolished by CXCR4 antagonists. TC14012 and TN14003 antagonized the antiapoptotic effect of synthetic CXCL12 and stromal cell-mediated protection of CLL cells from spontaneous apoptosis. Furthermore, we found that stromal cells protected CLL cells from chemotherapy-induced apoptosis. Treatment with CXCR4 antagonists resensitized CLL cells cultured with stromal cells to fludarabine-induced apoptosis. These findings demonstrate that CXCR4 blocking agents effectively antagonize CXCL12-induced migratory and signaling responses and stromal protection of CLL cells from spontaneous or fludarabine-induced apoptosis. As such, small molecular CXCR4 antagonists may have activity in the treatment of patients with this disease. (Blood. 2005;106:1824-1830)

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Importance of PI3 Kinase Family for Crosstalk between Chronic Lymphocytic Leukemia B Cells and the Stromal Microenvironment: Therapeutic Implications.

Blood ◽

10.1182/blood.v110.11.1125.1125 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1125-1125

Author(s):

Matthias T.W. Niedermeier ◽

Justyna Rawluk ◽

Zachary Knight ◽

Kevan Shokat ◽

William G. Wierda ◽

...

Keyword(s):

Cell Migration ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Stromal Cells ◽

Therapeutic Potential ◽

Dominant Role ◽

Lymphocytic Leukemia ◽

Drug Induced ◽

Cxcr4 Signaling ◽

Induced Apoptosis

Abstract Nontumoral accessory cells such as marrow stromal cells (MSC) or nurselike cells (NLC), which constitute the leukemia microenvironment, constitutively secrete the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). CXCL12 transduces signals via its receptor CXCR4, which is expressed at high levels by Chronic Lymphocytic Leukemia (CLL) B cells. Via the CXCL12-CXCR4 axis, CLL cells migrate and adhere to stromal cells. Adhesion to stromal cells protects CLL cells from spontaneous and drug-induced apoptosis in a contact-dependent fashion. Signaling pathways regulating these processes in CLL B cells are largely unknown. Here, we examined the importance of phosphatidyl-inositide 3-kinases (PI3-K) for migration and viability of CLL B cells using non-specific and isoform-specific PI3-K inhibitors. The importance of PI3-K for migration of CLL cells to CXCL12 was determined by transwell chemotaxis and pseudoemperipolesis (PEP) assays. Inhibition of PI3-K resulted in a significant reduction of CLL cell migration in chemotaxis and PEP assays. In comparison to untreated CLL cells, Ly 294002 inhibited chemotaxis to 65 ± 4.6% of untreated controls. Using a panel of isoform-specific PI 3-K inhibitors (PI-103, PIK-90, IC87114, TGX-115, ZK-75), we observed inhibition of chemotaxis by the multi-targeted compounds PI-103 (51.4 ± 0.2%) and PIK-90 (57.5 ± 8.9%), whereas p110beta and delta inhibition had no effect. We conclude from this part of the study that PI3-kinases play an important role for CXCR4 signaling in CLL B cells, mediating migratory responses and protection from apoptosis. Experiments with inhibitors of PI3-K with higher target selectivity suggest a dominant role for the class I PI3-K p110alpha for migration in response to CXCL12. Because adhesion to stroma mediates protection from chemotherapeutic drugs, we tested PI3-K inhibitors alone and in combination with fludarabine in CLL-stroma co-cultures. Pre-treatment of CLL cells with the PI3-K inhibitors Ly 294002, PI-103, and PIK-90 resulted in a significant decrease in viability of CLL cells co-cultured with and without stroma. Moreover, PI3-K isoform specific inhibitors enhance the cytotoxicity of Fludarabine and partially reverse the protective effect of stromal cells on fludarabine-induced apoptosis. Collectively, this study establishes that PI3-Ks play an important role in CXCR4 signaling for CLL cell migration and adhesion to stromal cells. New, isoform-specific PI3-K inhibitors enhance the cytotoxicity of fludarabine in suspension cultures and in co-cultures with stromal cells. Therefore, the therapeutic potential of PI3-K inhibitors alone or in combination with fludarabine should further be investigated. Figure. Figure.

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Phase I to II Multicenter Study of Oblimersen Sodium, a Bcl-2 Antisense Oligonucleotide, in Patients With Advanced Chronic Lymphocytic Leukemia

Journal of Clinical Oncology ◽

10.1200/jco.2005.02.4364 ◽

2005 ◽

Vol 23 (30) ◽

pp. 7697-7702 ◽

Cited By ~ 142

Author(s):

Susan M. O'Brien ◽

Charles C. Cunningham ◽

Anatoliy K. Golenkov ◽

Anna G. Turkina ◽

Steven C. Novick ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Phase I ◽

Phase Ii ◽

Intravenous Infusion ◽

Single Agent ◽

Plasma Concentrations ◽

Parent Drug ◽

Lymphocytic Leukemia ◽

Complete Disappearance ◽

Continuous Intravenous Infusion

Purpose To determine the maximum-tolerated dose (MTD), efficacy, safety, and pharmacokinetics of oblimersen sodium in patients with advanced chronic lymphocytic leukemia (CLL). Patients and Methods Eligible patients had relapsed or refractory CLL after treatment with fludarabine. Oblimersen was administered at doses ranging from 3 to 7 mg/kg/d as a 5-day continuous intravenous infusion in cycle 1 and as a 7-day continuous intravenous infusion in subsequent cycles every 3 weeks in stable or responding patients. Results Forty patients were enrolled and treated (14 patients in phase I and 26 patients in phase II). Dose-limiting reactions in phase I included hypotension and fever, and the MTD for phase II dosing was established at 3 mg/kg/d. Two (8%) of 26 assessable patients achieved a partial response. Other evidence of antitumor activity included ≥ 50% reduction in splenomegaly (seven of 17 patients; 41%), complete disappearance of hepatomegaly (two of seven patients; 29%), ≥ 50% reduction of lymphadenopathy (seven of 22 patients; 32%), and ≥ 50% reduction in circulating lymphocyte counts (11 of 22 patients; 50%). Adverse events included transient hypotension, fever, fatigue, night sweats, diarrhea, nausea, vomiting, hypokalemia, and cough. Plasma concentrations of oblimersen (parent drug) and its major metabolites were variable. Renal clearance represented only a small portion of total parent drug clearance. Conclusion Dosing with oblimersen sodium in patients with CLL is limited by development of a cytokine release syndrome that is characterized by fever, hypotension, and back pain. Oblimersen sodium has modest single-agent activity in heavily pretreated patients with advanced CLL, and further evaluation of its activity in combination with cytotoxic drugs is warranted.

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