Apoptosis In Chronic Lymphocytic Leukemia Research Articles

VSV Oncolysis in Combination With the BCL-2 Inhibitor Obatoclax Overcomes Apoptosis Resistance in Chronic Lymphocytic Leukemia

In chronic lymphocytic leukemia (CLL), overexpression of antiapoptotic B-cell leukemia/lymphoma 2 (BCL-2) family members contributes to leukemogenesis by interfering with apoptosis; BCL-2 expression also impairs vesicular stomatitis virus (VSV)-mediated oncolysis of primary CLL cells. In the effort to reverse resistance to VSV-mediated oncolysis, we combined VSV with obatoclax (GX15-070)—a small-molecule BCL-2 inhibitor currently in phase 2 clinical trials—and examined the molecular mechanisms governing the in vitro and in vivo antitumor efficiency of combining the two agents. In combination with VSV, obatoclax synergistically induced cell death in primary CLL samples and reduced tumor growth in severe combined immunodeficient (SCID) mice-bearing A20 lymphoma tumors. Mechanistically, the combination stimulated the mitochondrial apoptotic pathway, as reflected by caspase-3 and -9 cleavage, cytochrome c release and BAX translocation. Combination treatment triggered the release of BAX from BCL-2 and myeloid cell leukemia-1 (MCL-1) from BAK, whereas VSV infection induced NOXA expression and increased the formation of a novel BAX-NOXA heterodimer. Finally, NOXA was identified as an important inducer of VSV-obatoclax driven apoptosis via knockdown and overexpression of NOXA. These studies offer insight into the synergy between small-molecule BCL-2 inhibitors such as obatoclax and VSV as a combination strategy to overcome apoptosis resistance in CLL.

Novel X‐linked inhibitor of apoptosis inhibiting compound as sensitizer for TRAIL‐mediated apoptosis in chronic lymphocytic leukaemia with poor prognosis

Given that aggressive DNA damaging chemotherapy shows suboptimal efficacy in chronic lymphocytic leukaemia (CLL), alternative therapeutic approaches are needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce tumour-specific apoptosis. However, apoptosis might be inhibited by elevated levels of X-linked inhibitor of apoptosis (XIAP). Use of XIAP-inhibiting compounds might sensitize primary CLL cells towards TRAIL-mediated apoptosis. A novel small molecule, compound A (CA), an inhibitor of XIAP, was used in combination with TRAIL to induce apoptosis in primary CLL cells (n = 48). XIAP was significantly more highly expressed in primary CLL cells (n = 28) compared to healthy B cells (n = 16) (P = 0·02). Our data obtained by specific knock-down of XIAP by siRNA identified XIAP as the key factor conferring resistance to TRAIL in CLL. Combined treatment with CA/TRAIL significantly increased apoptosis compared to untreated (P = 8·5 × 10⁻¹⁰), solely CA (P = 4·1 × 10⁻¹²) or TRAIL treated (P = 4·8 × 10⁻¹⁰) CLL cells. CA rendered 40 of 48 (83·3%) primary CLL samples susceptible to TRAIL-mediated apoptosis. In particular, cells derived from patients with poor prognosis CLL (ZAP-70(+) , IGHV unmutated, 17p-) were highly responsive to this drug combination. Our highly-effective XIAP inhibitor CA, in concert with TRAIL, shows potential for the treatment of CLL cases with poor prognosis and therefore warrants further clinical investigation.

PDE4 Inhibitors Enhance DNA Damage-Induced Apoptosis In CLL

AbstractAbstract 2902Treatment of primary chronic lymphocytic leukemia (CLL) cells with PDE4 inhibitors induces apoptosis while comparable treatment of primary human T and B cells from healthy donors does not. PDE4 inhibitor treatment also augments glucocorticoid-induced apoptosis in CLL cells. We now report that PDE4 inhibitor treatment enhances CLL apoptosis induced by DNA damage. Using drug combination analysis, treatment with etoposide and rolipram resulted in a greater than additive apoptotic effect than predicted by a Loewe additivity model at many dose combinations. In order to directly address the role of DNA damage and rule out effects on drug uptake and efflux, we next examined the ability of PDE4 inhibitors to modulate gamma-irradiation-induced apoptosis in CLL. While most patients demonstrated in vitro sensitivity to rolipram and gamma irradiation, a small subset of patients, including those known to be p53 mutant, that lacked a substantial response to ionizing radiation also lacked sensitivity to rolipram (p < 0.01). Those patients with intact sensitivity to radiation, demonstrated at least an additive, or greater-than-additive at some doses, effect on apoptosis when PDE4 inhibitor treatment was combined with gamma irradiation treatment (p < 0.05 at 1 Gy and 20 mM rolipram). As judged by comet assays, gamma radiation-induced DNA double stranded breaks were repaired within one hour of induction regardless of exposure to rolipram. As judged by Western analysis and flow cytometry, the kinetics of gamma irradiation-induced H2AX phosphorylation, a marker for DNA damage, were not altered by addition of rolipram in a manner that correlated with apoptosis. Finally, rolipram treatment did not further augment levels of p53, p21cip, or phospho-Chk2. These results suggest that PDE4 inhibitors did not alter double strand DNA break repair nor alter upstream radiation-induced p53-mediated signaling events. As PDE4 inhibitors are known to induce CLL apoptosis through a mitochondrial pathway, our current studies are focused on identifying the mechanism by which DNA damage and PDE4 inhibitor signaling pathways converge at the mitochondrion to enhance apoptosis. Regardless of the precise mechanism by which these supra-additive apoptotic effects occur, our study adds credence to the concept that PDE4 inhibitors may prove to be important sensitizing adjuvants in the treatment of human B cell malignancies. Disclosures:No relevant conflicts of interest to declare.

Reducing Mcl-1 by Homoharringtonine Induces Apoptosis In Chronic Lymphocytic Leukemia Cells

AbstractAbstract 2896Homoharringtonine (HHT, omacetaxine) is an inhibitor of translation elongation that is currently in clinical evaluation in chronic myelogenous leukemia. We hypothesized that this compound would be active in inducing apoptosis in chronic lymphocytic leukemia (CLL) based on the following rationale: 1): CLL cells are characterized by their dependence on the overexpression of anti-apoptotic proteins to maintain their survival; 2): one of the key anti-apoptotic proteins, Mcl-1, turns over rapidly. Thus, upon transient exposure to a translation inhibitor, Mcl-1 expression would decrease rapidly and allow the pro-apoptotic proteins to activate the mitochondria-dependent apoptosis. Using tritiated leucine incorporation as an indicator of new protein synthesis, our results in primary CLL cells demonstrated that HHT was more potent at inhibiting translation than cycloheximide. A polysome profiling analysis confirmed that Mcl-1 translation was blocked by HHT. Mcl-1 protein levels were reduced significantly after HHT exposure, measured by an immunoblotting analysis. This led to mitochondrial membrane depolarization and initiation of apoptosis, analyzed by flow cytometry. Sensitivity to HHT varied in different CLL samples. Further studies showed that this heterogeneous response was independent of the CLL prognostic factors such as the mutation status of the IgVH or ZAP70 expression. Rather, it correlated significantly to the baseline Mcl-1 expression in each CLL sample and the extent of reduction of Mcl-1 protein. A detailed time course study showed that Mcl-1 reduction was evident as early as 2 h and continue to decrease in the next 6 to 8 h; cell death started in 2 h and continued to increase for 24-h. The reduction of Mcl-1 was not affected by the pan caspase inhibitor zVAD, indicating that reduction of Mcl-1 was the initiation event of apoptosis, rather than the results of caspase cleavage. On the contrary, the proteosome inhibitor MG-132 caused retention of the Mcl-1 level in the presence of HHT, suggesting that when Mcl-1 synthesis was blocked by HHT, the remaining Mcl-1 in the cells was degraded rapidly in a proteosome-dependent mechanism. As the action of HHT relied largely on the rapid turn-over of Mcl-1 protein, blocking Mcl-1 degradation by the proteosome inhibitor would counteract the effect of HHT. This explained the observed antagonistic reaction of HHT and MG-132 when they were used together. Further, interaction of CLL cells with the microenvironment has been shown to protect CLL cells from chemotherapy. Incubating CLL cells on either a murine or human stromal cell layer in vitro clearly protected CLL cells from the toxicity of fludarabine. There was an induction of both Mcl-1 transcript and protein levels, which could explain this protection. However, this induction was reversed by HHT, overcoming stromal cell-mediated protection. Finally, because of the important role of Mcl-1 in the pathogenesis CLL, we hypothesized a sequential blockade strategy that inhibits both Mcl-1 transcription and translation to completely block Mcl-1 synthesis. Median effect analysis showed that combining the transcription inhibitor SNS-032 together with HHT killed the CLL cells synergistically. An immunoblotting analysis demonstrated that the combination of the two drugs depleted Mcl-1 more completely than either single drug at the same concentration, which correlated to a greater induction of apoptosis than either drug alone. Taken together, our data demonstrated that transiently inhibiting translation and depleting Mcl-1 was the major mechanism of HHT-induced apoptosis in CLL cells. This action overcame stromal cell-mediated protection and was synergistic with inhibitors of transcription. Thus, these data provided rationale for the clinical development of HHT in CLL, and for the design of combination strategies. Disclosures:No relevant conflicts of interest to declare.

Effect of Valproic Acid (VPA) on Fludarabine Activity In Chronic Lymphocytic Leukemia (CLL) Patients

AbstractAbstract 4626Valproic acid (VPA) is a first-generation anti-epileptic drug that has been shown to inhibit proliferation and induce apoptosis in various hematological malignancies, including multiple myeloma and chronic lymphocytic leukemia (CLL). VPA-induced apoptosis is thought to be due to its ability to inhibit histone deacetylases (HDACs), resulting in hyper-acetylation of histones and altered expression levels of pro- and anti-apoptotic genes. We show here that the mechanism of VPA-induced apoptosis in CLL is associated with activation of the TNF-related apoptosis-inducing ligand (TRAIL) apoptotic pathway in vitro and ex vivo. TRAIL induces cell death by binding to death receptors 4 and 5 (DR4, DR5), which in turn activate the extrinsic apoptotic pathway. We have previously shown that 30–40% of the activities of fludarabine and chlorambucil are mediated through the TRAIL pathway, and this is related to up-regulation of the expressions of DR4 and DR5. We confirm here that VPA induces apoptosis in two CLL-like cell lines, I-83 and BJAB. Co-treatment of the two cell lines with VPA and activating antibodies against either DR4 (ETR1) or DR5 (ETR2) enhanced VPA-induced apoptosis in both cell lines after 24 hours. Conversely, blocking the TRAIL apoptotic pathway with DR4:FC fusion protein decreased the cytotoxicity of VPA, as well as another HDAC inhibitor, trichostatin A, in BJAB cells. These results confirmed the importance of activation of the TRAIL apoptotic pathway for the antitumor activity of VPA. Treatment with VPA also induced formation of reactive oxygen species (ROS) and enhanced fludarabine-induced apoptosis in cell lines and primary CLL cells. Having shown the effects of VPA in vitro, a phase II clinical trial was initiated at CancerCare Manitoba to determine the activity of VPA in CLL, either when used alone or in combination with fludarabine. Five patients who had received at least one prior therapy with fludarabine have to date been examined. Three out of 5 patients were fludarabine-resistant, as defined as no response to fludarabine or relapse < 6-months after completion of treatment with fludarabine. Patients received VPA at a starting dose of 15 mg/kg/day orally in divided doses, with the goal of reaching a serum level of >1 mM. Various side effects were observed with higher doses of VPA, including anaemia, thrombocytopaenia, GI toxicity and fatigue. No responses were seen after 28 days using VPA alone. However, in four patients who continued on VPA with fludarabine, 3 patients showed a >50% fall in lymphocyte/lymph node size after receiving 5 cycles of the combination. Peripheral blood samples were obtained prior to each treatment cycle, and mononuclear cells were isolated for analysis by immunoblotting and immunocytochemistry. Levels of both histone 3-acetyl and histone 4-acetyl initially increased and then fluctuated during the course of treatment. When DR4 and DR5 levels were examined by immunoblotting, DR4 levels increased during the course of therapy, while no significant changes in DR5 levels were observed. Expression of mRNA levels of genes involved in apoptosis was examined in one patient before and 28 days following VPA. There was a global up-regulation of both pro- and anti-apoptotic genes (as categorized by their gene ontology) which likely explains the effect of VPA on fludarabine sensitivity in CLL. In summary, VPA induces hyper-acetylation of histones in CLL and activates the TRAIL apoptotic pathway through upregulation of DR4 levels. While VPA is ineffective as a single agent in CLL, it can sensitize CLL cells to fludarabine and could be used as an adjuvant in fludarabine-based treatment regimens. Ongoing studies are evaluating the effectiveness of lower and less toxic doses of VPA in combination with fludarabine in CLL. Disclosures:Off Label Use: Valproic acid (VPA). VPA is being tested as a possible adjunct in treating chronic lymphocytic leukemia.

Novel X-Linked Inhibitor of Apoptosis (XIAP) Inhibiting Compound as Sensitizer for TRAIL-Mediated Apoptosis In Chronic Lymphocytic Leukemia with Poor Prognosis

AbstractAbstract 1375 Background:Since aggressive DNA damaging chemotherapy shows suboptimal efficacy in chronic lymphocytic leukemia (CLL), alternative therapeutic approaches are needed. Moreover, there is an essential need to improve specific therapeutic regimes for “non-fit” patients, which cannot receive myeloablative therapies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce tumor-specific apoptosis. However, apoptosis might be inhibited by elevated X-linked inhibitor of apoptosis (XIAP) level, the only cellular protein capable to bind to and effectively inhibit caspases. Use of XIAP-inhibiting compounds might sensitize primary CLL cells towards TRAIL-induced lysis. Experimental design:We compared XIAP protein levels between freshly purified CD5+CD19+ primary CLL cells (n=28) and CD19+ B cells from healthy donors (n=16) by western blotting. In a knockdown approach, specific siRNAs against XIAP were nucleofected to check whether XIAP expression prevents TRAIL-mediated apoptosis in CLL. After proof of concept, we applied the novel small molecule IAP antagonizing compound (IAC), an inhibitor of XIAP, in combination with TRAIL to induce apoptosis in primary CLL cells (n=48). Compound A (CA) was developed based on the crystal structure of four amino acids of SMAC, which enabling SMAC to efficiently bind the BIR3 domain of XIAP. In contrast to the active compound CA, which consists of an amino terminal methyl alanine, the inactive compound CB used in our studies as a negative control has an amino terminal methyl glycine. This specific substitution results in a significant reduction of IAP binding capability of CB as CA has binding affinity to XIAP in the picomolar range and CB is a weak binder with micromolar binding affinity to XIAP. Results:XIAP is significantly higher expressed in primary CLL cells (n=28) compared to healthy B cells (n=16) (P=0.02). Our data obtained by specific knockdown of XIAP via siRNA identified XIAP as the key factor conferring resistance to TRAIL in CLL. Based on these results we used IAC in combination with TRAIL. Combined treatment with both drugs significantly increased apoptosis compared to untreated (P=8.5×10-10), solely IAC (P=4.1×10-12) or TRAIL treated (P=4.8×10-10) CLL cells. As a potent cellular caspase inhibitor, we also examined the involvement of caspases in CA/TRAIL-mediated apoptosis. Not surprisingly, co-application of pan-caspase inhibitor zVAD.fmk inhibited cell death induced by CA/TRAIL underscoring the apoptotic caspase-dependent cytotoxicity of CA/TRAIL treatment in CLL cells. IAC rendered 40 of 48 (83.3%) primary CLL samples susceptible towards TRAIL-mediated apoptosis. Especially cells derived from patients with poor prognosis (ZAP-70+, IGHV unmutated, 17p-) were highly responsive to this drug combination. Furthermore, this study reveals that TRAIL application alone induces apoptosis in poor-prognosis CLL samples (13,8% in ZAP-70+ (n=10) vs. 2,3 in ZAP-70- (n=9); P=0.0008), which correlates with the elevated expression levels of TRAIL-R1 and –R2 on ZAP-70+ CLL cells. To assess whether TRAIL treatment is CLL cell specific, healthy B cells (n=4) were exposed to TRAIL alone or CA(CB)/TRAIL and showed significantly lower susceptibility towards CA/TRAIL administration than CLL cells. Conclusion:XIAP is over-expressed in CLL and displays a suitable target to induce TRAIL-mediated apoptosis. The novel XIAP inhibitor used in our study was able to inhibit XIAP function at a concentration of 0,1μM. CA/TRAIL administration was also shown not to induce apoptosis in healthy donor B cells and might therefore also display an attractive option for “non-fit” CLL patients. Our highly effective XIAP inhibitor CA, in concert with TRAIL, shows potential for treatment of CLL of those cases with poor prognosis and therefore warrants further clinical investigation. Disclosures:No relevant conflicts of interest to declare.

AICAR Induces Apoptosis In B Lymphocytes From Balb/c Mice and Its Effect In Chronic Lymphocytic Leukemia Cells Is Synergic or Additive with Current Chemotherapy

AbstractAbstract 1815AICAR (5-aminoimidazole-4-carboxamide riboside or acadesine) induces apoptosis in chronic lymphocytic leukemia (CLL) cells, without affecting primary T lymphocytes (CampaÌ€s et al, Bood 2003). Available treatments for this disease generally induce remission, although nearly all patients relapse and CLL remains incurable. Thus, AICAR is a promising drug for the treatment of this B-cell neoplasm. It has been recently published that AICAR induces apoptosis by a p53- and AMPK-independent mechanism through upregulation of BIM and NOXA in CLL cells (SantidriaÃŒn&GonzaÃŒlez-GironeÌ€s et al, Blood in press). A clinical phase I/II study of AICAR is currently being conducted in CLL patients (http://clinicaltrials.gov/ct2/NCT00559624). This clinical study has shown that AICAR plasmatic levels in the micro molar range are achievable and safe when CLL patients are treated with the drug. In vivo assays were performed in mice to analyze the effects of AICAR on the peripheral lymphocyte population. Thus, 0.5 mg/g AICAR was administered intraperitoneally to Balb/c mice every 12 hours and every day blood was collected from the tail of treated (n = 3) and untreated (n = 3) mice. Significant differences (p< .05) were observed between control and treated groups in the number of lymphocytes from day 3 to 6 while the number of total leucocytes did not change. From day 4 the peripheral lymphocyte population started to recover and mice were sacrificed at day 8. In addition, AICAR induced a significant reduction (p< .05) on the percentage of B cells from day 3 to 5. Therefore, AICAR is effective in vivo decreasing the number of peripheral B lymphocytes. From the therapeutic point of view, it is interesting to analyze whether AICAR could synergize with the cytotoxic activity of the current chemotherapy used in CLL patients. Thus, cells from CLL patients (n = 4) were treated with AICAR (0.125, 0.25, 0.5 and 1 mM) and/or dexamethasone (1, 2.5, 5 and 10 μM), fludarabine (0.3, 0.6, 1.5 and 3 μM), chlorambucil (1.25, 2.5, 5 and 10 μM), mafosfamide (the active metabolite of cyclophosphamide) (0.25, 0.5, 1 and 2 μg/mL), rituximab (10, 25, 50 and 100 μg/mL) or alemtuzumab (1.25, 2.5, 5 and 10 μg/mL). The cytotoxic effect of the alkylating agents chlorambucil and mafosfamide was synergic or additive with the effect of AICAR in CLL cells depending on the concentration used of both drugs. The glucocorticoid dexamethasone synergized with AICAR in half of the samples analyzed. As for the nucleoside analogue fludarabine and the monoclonal antibodies rituximab and alemtuzumab, their apoptotic effect was additive with AICAR at some concentrations. Together, AICAR induces apoptosis in B lymphocytes from Balb/c mice when administered intraperitoneally and its cytotoxic effect in CLL cells is synergic or additive with the most common chemotherapy used in CLL treatment. Disclosures:de Frias:Advancell: Employment. Campàs:Advancell: Employment.

FK506 Binding Proteins as Targets in Anticancer Therapy

FK506 binding proteins (FKBPs) are the intracellular ligands of FK506 and rapamycin, two natural compounds with powerful and clinically efficient immunosuppressive activity. In recent decades, a relevant role for immunosuppressants as anticancer agents has emerged. Especially, rapamycin and its derivatives are used, with successful results, across a variety of tumors. Of note, rapamycin and FK506 bind to FKBP12, and the resulting complexes interfere with distinct intracellular signaling pathways driven, respectively, by the mammalian target of rapamycin and calcineurin phosphatase. These pathways are related to T-cell activation and growth. Hyperactivation of the mammalian target of rapamycin (mTOR), particularly in cancers that have lost the tumor suppressor gene PTEN, plays an important pathogenetic role in tumor transformation and growth. The signaling pathway involving calcineurin and nuclear factors of activated T-lymphocytes is also involved in the pathogenesis of different cancer types and in tumor metastasis, providing a rationale for use of FK506 in anticancer therapy. Recent studies have focused on FKBPs in apoptosis regulation: Targeting of FKBP12 promotes apoptosis in chronic lymphocytic leukemia, FKBP38 knockdown sensitizes hepatoma cells to apoptosis, and FKBP51 silencing overcomes resistance to apoptosis in acute lymphoblastic leukemia, prostate cancer, melanoma, and glioma. Interestingly, derivatives of FK506 that have the same FKBP12-binding properties as FK506 but lack functional immunosuppressant activity, exert the same apoptotic effect as FK506 in chronic lymphocytic leukemia.These findings suggest that a direct FKBP inhibition represents a further mechanism of immunosuppressants.' anticancer activity. In this review, we focus on the role of FKBP members in apoptosis control and summarize the data on the antitumor effect of selective targeting of FKBP.

AICAR induces apoptosis independently of AMPK and p53 through up-regulation of the BH3-only proteins BIM and NOXA in chronic lymphocytic leukemia cells

Abstract5-Aminoimidazole-4-carboxamide riboside or acadesine (AICAR) induces apoptosis in chronic lymphocytic leukemia (CLL) cells. A clinical study of AICAR is currently being performed in patients with this disease. Here, we have analyzed the mechanisms involved in AICAR-induced apoptosis in CLL cells in which it activates its only well-known molecular target, adenosine monophosphate-activated protein kinase (AMPK). However, AMPK activation with phenformin or A-769662 failed to induce apoptosis in CLL cells and AICAR also potently induced apoptosis in B lymphocytes from Ampkα1−/− mice, demonstrating an AMPK-independent mechanism of cell death. Importantly, AICAR induced apoptosis irrespective of the tumor suppressor TP53 or ataxia telangiectasia mutated (ATM) status via induction of the mitochondrial pathway. Apoptosis was preceded by an increase in mRNA and protein levels of proapoptotic BCL-2 family proteins of the BH3-only subgroup, including BIM, NOXA, and PUMA in CLL cells. Strikingly, B lymphocytes from Noxa−/− or Bim−/− mice were partially protected from the cytotoxic effects of AICAR. Consistently, B cells from Noxa−/−/Bim−/− mice resisted induction of apoptosis by AICAR as potently as B lymphocytes overexpressing transgenic BCL-2. These findings support the notion that AICAR is an interesting alternative therapeutic option for CLL patients with impaired p53 function and resistance to conventional chemotherapy.

Targeting Mitochondrial Glutaminase Activity Inhibits Oncogenic Transformation

Rho GTPases impact a number of activities important for oncogenesis. We describe a small molecule inhibitor that blocks oncogenic transformation induced by various Rho GTPases in fibroblasts, and the growth of human breast cancer and B lymphoma cells, without affecting normal cells. We identify the target of this inhibitor to be the metabolic enzyme glutaminase, which catalyzes the hydrolysis of glutamine to glutamate. We show that transformed fibroblasts and breast cancer cells exhibit elevated glutaminase activity that is dependent on Rho GTPases and NF-κB activity, and is blocked by the small molecule inhibitor. These findings highlight a previously unappreciated connection between Rho GTPase activation and cellular metabolism and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.

Resveratrol increases rate of apoptosis caused by purine analogues in malignant lymphocytes of chronic lymphocytic leukemia

In this study, we attempted to assess the interactions of resveratrol, a natural compound present in various plant species, with the purine analogues fludarabine and cladribine in terms of their effects on DNA damage and apoptosis in chronic lymphocytic leukemia (CLL) cells. The experiments were performed ex vivo using short-term cell cultures of blood and bone marrow cells from newly diagnosed untreated patients. We analyzed the expression of active caspase-3 and the BCL-2/BAX ratio as markers of apoptosis and the expression of phosphorylated histone H2AX (γH2AX) and activated ATM kinase, which are reporters of DNA damage. The results of our study revealed that resveratrol induced apoptosis in CLL cells in a tumor-specific manner but did not affect non-leukemic cells, and apoptosis was associated with a decreased BCL2/BAX ratio. Here, we report for the first time that both resveratrol + fludarabine and resveratrol + cladribine caused a higher rate of apoptosis in comparison to the rate caused by a single drug. The percentage of apoptotic cells induced by resveratrol alone was higher in the group of patients with better prognostic markers than in those with worse prognostic markers. However, the rates of apoptosis caused by resveratrol combined with purine analogues were independent of ZAP-70 and CD38 expression and the clinical state of the disease; they were only dependent on the presence of high-risk cytogenetic abnormalities. We also observed an increase in γH2AX expression together with a rise in activated ATM in most of the analyzed samples. The obtained results indicate that resveratrol might warrant further study as a new therapeutic option for CLL patients. This naturally occurring substance may be used as a single agent, especially in older persons for whom there are some limitations for the use of aggressive treatment. On the other hand, a lower purine analogue dose could potentially be used in combination with resveratrol because of their combined effect. One of the mechanisms of action of resveratrol is the induction of DNA damage, which ultimately leads to apoptosis.

17-DMAG targets the nuclear factor-κB family of proteins to induce apoptosis in chronic lymphocytic leukemia: clinical implications of HSP90 inhibition

AbstractThe HSP90 client chaperone interaction stabilizes several important enzymes and antiapoptotic proteins, and pharmacologic inhibition of HSP90 results in rapid client protein degradation. Therefore, HSP90 inhibition is an attractive therapeutic approach when this protein is active, a phenotype commonly observed in transformed but not normal cells. However, preclinical studies with HSP90 inhibitors such as 17-AAG demonstrated depletion of only a subset of client proteins and very modest tumor cytotoxicity in chronic lymphocytic leukemia (CLL) cells. Herein, we describe another HSP90 inhibitor, 17-DMAG, which is cytotoxic to CLL but not normal lymphocytes. Treatment with 17-DMAG leads to depletion of the HSP90 client protein IKK, resulting in diminished NF-κB p50/p65 DNA binding, decreased NF-κB target gene transcription, and caspase-dependent apoptosis. Furthermore, treatment with 17-DMAG significantly decreased the white blood cell count and prolonged the survival in a TCL1-SCID transplant mouse model. The ability of 17-DMAG to function as an NF-κB inhibitor is of great interest clinically, as few currently available CLL drugs target this transcription factor. Therefore, the effect of 17-DMAG on NF-κB signaling pathways represents a novel therapy warranting further clinical pursuit in this and other B-cell lymphoproliferative disorders.

209 Nicotinamide acts as pharmacological agent by promoting apoptosis in chronic lymphocytic leukemia through the activation of the p53/miR-34a/SIRT1 tumour suppressor network

209 Nicotinamide acts as pharmacological agent by promoting apoptosis in chronic lymphocytic leukemia through the activation of the p53/miR-34a/SIRT1 tumour suppressor network

Abstract 4508: Targeting XIAP as a novel paradigm to induce apoptosis in chronic lymphocytic leukemia

Abstract Evasion of apoptosis is a hallmark of chronic lymphocytic leukemia (CLL), calling for new concepts to overcome apoptosis resistance. We therefore investigated whether targeting the antiapoptotic protein XIAP by small molecule inhibitors would sensitize for apoptosis. Here, we provide first evidence that XIAP inhibitors in combination with the death receptor ligand TRAIL present a new strategy to synergistically trigger apoptosis in CLL even in subgroups with resistant disease. Analysis of apoptosis regulatory proteins reveals that XIAP, cIAP1 and cIAP2 are expressed at high levels in primary CLL samples. Proofs of concept studies in CLL cell lines demonstrate that subtoxic concentrations of several distinct XIAP inhibitors significantly enhance TRAIL-induced apoptosis. By comparison, no sensitization for death receptor-induced apoptosis is observed in the presence of a structurally related control compound that only weakly binds to XIAP, demonstrating the specificity of the sensitization effect of XIAP inhibitors. Importantly also in primary CLL samples, subtoxic concentrations of XIAP inhibitors act in concert with TRAIL to trigger apoptosis in 18 of 27 cases (67%), whereas primary CLL cells are resistant to treatment with TRAIL alone. Analysis of combination index reveals that this interaction of XIAP inhibitor and TRAIL is highly synergistic. Mechanistic studies in primary CLL cells show that the addition of XIAP inhibitor profoundly enhances TRAIL-induced cleavage of caspase-3 into active fragments and significantly increases caspase-3 enzymatic activity. The broad range caspase inhibitor zVAD. fmk completely blocks apoptosis in response to combination treatment with XIAP inhibitor and TRAIL, pointing to caspase-dependent apoptosis. Intriguingly, the cooperative interaction of XIAP inhibitor and TRAIL is even evident in several distinct subgroups of CLL patients with poor prognostic features, including patients with 17p deletion, TP53 mutation, chemotherapy-refractory disease or unmutated VH genes. Interestingly, we found that cases with unmutated VH genes are significantly more sensitive to XIAP inhibitor- and TRAIL-induced apoptosis compared to VH gene mutated samples, pointing to a role of B-cell receptor signaling in the regulation of apoptosis in CLL cells. In conclusion, we provide first evidence that XIAP inhibitors in combination with TRAIL present a novel strategy to trigger apoptosis even in resistant forms and poor prognostic subgroups of CLL. These findings have important implications for the development of innovative approaches to overcome the intrinsic resistance to apoptosis in CLL. Since IAP inhibitors as well as TRAIL receptor agonists as single agents are currently already under evaluation in early clinical trials, it is feasible that such combination protocols of XIAP inhibitors and TRAIL could be translated into clinical application in CLL. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4508.

Abstract 4431: A novel derivative of the Cdk inhibitor roscovitine that induces apoptosis in CLL and overcomes stromal cell-mediated protection

Abstract Cmpd 5 is a derivative of R-roscovitine (CYC-202, seliciclib) with increased potency that selectively inhibits Cdk2, Cdk5 and Cdk9. In chronic lymphocytic leukemia (CLL) a disease that is addicted to the over-expression of anti-apoptotic proteins for survival, inhibition of Cdk9 by Cmpd 5 reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription. These actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1, and induced apoptosis in CLL cells in vitro. Cmpd 5 was about 30 times more potent than its parent compound R-roscovitine with an IC50 of 0.86 µM after a 24-hr incubation. Although cell death was initiated after a 4-hr incubation in a time course study of Cmpd 5-induced apoptosis, the maximum cell death was not reached until 10-12 hr. In addition, it was known that co-culture of CLL cells with the marrow and lymphoid stromal cells may be responsible for resistance to fludarabine therapy. We evaluated Cmpd 5 in overcoming such stromal cell-mediated protection. Cmpd 5 killed the CLL cells similarly in the presence or absence of the human stromal cell line StromaNKtert without toxicity to the stromal cells, whereas the stromal cells clearly protected the CLL cells from the toxicity of fludarabine. Since Mcl-1 is the major target of Cmpd 5 in CLL, and the action of Cmpd 5 relies on the intrinsically rapid turn-over rate of Mcl-1, we compared the protein levels and the half-life of Mcl-1 in CLL cells in the presence or absence of the stromal cell layer. After an overnight incubation of CLL cells with the StromaNKtert cells, there was a 3-4 fold induction of Mcl-1 transcript and protein. However, there was no difference in the Cmpd 5-induced decay rate of either the Mcl-1 mRNA or protein, indicating that stromal cells did not alter the stability of Mcl-1. Therefore, the elevated Mcl-1 level likely indicated increased biosynthesis, rather than enhanced stability. Thus, the activation of the survival pathways and induction of the biosynthesis of Mcl-1 by the stromal cells may protect CLL cells from fludarabine-induced apoptosis but loss of the short-lived anti-apoptotic proteins was not affected, explaining the lack of stromal cell protection for CLL cells from Cmpd 5. Similar time-dependence of cell death induction as well as stromal cell response was also observed with other inhibitors of transcription including flavopiridol, R-roscovitine and Actinomycin D, implying a class effect. Taken together, our data suggested that Cmpd 5 is a promising candidate for clinical development for CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4431.

Abstract 228: Cucurbitacin-I (JSI-124) induces apoptosis in chronic lymphocytic leukemia (CLL) cells through inhibiting STAT3 activation and XIAP expression

Abstract Abstract AACR April-2010 Cucurbitacin-I (JSI-124) induces apoptosis in Chronic Lymphocytic Leukemia (CLL) cells through inhibiting STAT3 activation and XIAP expression. Ganchimeg Ishdorj, James B Johnston and Spencer B Gibson Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in North America. Current therapies control the disease but eventually the disease recurs and becomes drug resistant. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in cell survival and induces proliferation, whereas deletion of STAT3 results in cell death. Constitutive phosphorylation of STAT3 on tyrosine 705 residues has been found in a number of solid tumors and hematologic malignancies. Unlike other tumors, STAT3 is constitutively phosphorylated (p) on serine 727 but not on tyrosine residues in CLL. These observations lead to investigation of the effect of STAT3/JAK2 inhibitor Cucurbitacin-I (JSI-124) in CLL cells. We proposed that the targeting STAT3 phosphorylation may induce apoptosis in CLL cells. We studied B-cell lines (BJAB, I-83, NALM-6) and primary B-cells from patients with CLL using immunoprecipitation and found that JSI-124 suppressed serine pSTAT3 and induced apoptosis of these cells as determined by FACS analysis and membrane permeability assay. Using western blotting analysis, caspase-8 activation was observed in these cells after JSI-124 treatment. Since the activation of caspases are inhibited by members of the inhibitor of apoptosis (IAP) and Bcl-2 protein families and STAT3 has been shown to regulate their expression, we investigated anti-apoptotic members of these families. JSI-124 treatment led to downregulation of IAP protein XIAP, while Bcl-xL and Mcl-1 levels remained unchanged. In addition, JSI-124 selectively induced activation of JNK signaling pathways in CLL cells. Taken together, our results indicate that JSI-124 causes apoptosis through alterations in signal transduction pathways and could be a useful treatment in CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 228.

Abstract 3493: ON 01910. Na, a clinical trial stage multi-kinase inhibitor, induces apoptosis in chronic lymphocytic leukemia (CLL) cells through inhibition of PI3K/AKT and activation of the JNK pathway resulting in NOXA and BIM upregulation

Abstract Chronic Lymphocytic leukemia (CLL), the most common leukemia in Western countries, is a clinically heterogeneous disease characterized by the accumulation of mature B lymphocytes, where disease progression is driven by survival and proliferation signals provided by the tumor microenvironment. ON 01910. Na (Onconova Therapeutics) belongs to the family of styryl benzyl sulfones, a novel family of non-ATP competitive kinase inhibitors. ON 01910. Na is under clinical development in hematologic and solid tumors. Here we show that ON 01910. Na induced apoptosis in CLL samples (n=28) in vitro, without showing significant toxicity against T-cells or normal B-cells. Twenty four CLL samples were highly sensitive to ON 01910. Na (mean IC50=0.89 μM) a concentration readily achieved in phase I clinical trials, three showed moderate sensitivity (mean IC50=4.26 μM) and one was resistant (IC50 &amp;gt;8 μM). There were no significant differences in ON01910. Na cytotoxicity against CLL cells expressing mutated (mean IC50=1.33 μM) or unmutated (mean IC50 =1.22μM) IgVH sequences. ON 01910. Na was similarly effective against tumor cells carrying 17p or 11q deletions. ON 01910. Na activated Bax and Bak, leading to mitochondrial depolarization. To delineate the upstream pathways underlying ON 01910. Na induced apoptosis, we performed Gene Expression Profiling (GEP) in CLL cells treated in vitro for 4 and 10 hours with ON 01910. Na. Gene Set Enrichment Analysis (GSEA) identified gene sets indicating BCR and PI3K inhibition (FDR&amp;lt;0.2). In keeping with PI3K inhibition, we found that ON 19010. Na inhibited phosphorylation of Akt and Foxo3a, and FOXO3 target genes, including Bim, were upregulated. In addition, GSEA identified induction of AP-1 gene sets (FDR&amp;lt;0.01) suggesting the generation of reactive oxygen species (ROS) by ON 01910. Na. Consistently, we found a rapid increase in ROS in cells treated with ON 01910. Na and ROS scavengers protected cells from its cytotoxic effect. ROS upregulated the proapoptotic BH3-only protein Noxa, and knockdown of Noxa by shRNA decreased the sensitivity to the drug by 60%. Our results identify ON 01910. Na as a promising agent in the treatment of CLL with an interesting dual mechanism of action: inhibition of the PI3K/AKT survival pathway with Foxo mediated upregulation of BIM and the induction of oxidative stress resulting in Noxa upregulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3493.

Abstract 2993: Vinblastine induces very rapid apoptosis in chronic lymphocytic leukemia and mantle cell lymphoma

Abstract Chronic lymphocytic leukemia (CLL) and mantle cell lymphomas are initially responsive to traditional therapies, but invariably relapse and are generally considered incurable. CLL is caused by an accumulation of mature resting B cells, due to a defect in cellular apoptosis rather than increased cell division. Most CLL cells maintain high levels of the anti-apoptotic protein BCL2 which protects them from apoptotic death, suggesting that they may be sensitive to BCL2-targeting drugs. Freshly isolated lymphocytes from CLL patients were exposed to the BH3 mimetic ABT-737, targeting BCL2 and BCL-XL anti-apoptotic proteins. Comparable to previous reports, ABT-737 displayed acute concentration-dependent toxicity, with low levels of ABT-737 (10 nM) inducing apoptosis as early as 3 hours. ABT-737 sensitivity has been attributed to its ability to displace Bim from BCL2/BCL-XL leading to intrinsic apoptosis. Consistent with this mechanism, we report three mantle cell lymphoma cell lines that are deficient in Bim are resistant to ABT-737. However, many other cell lines containing Bim are also resistant to ABT-737. This resistance has been attributed to the presence of MCL-1; however the Bim-deficient Mino cell line has very low MCL-1 levels yet is still resistant to ABT-737. Surprisingly, the Bim-deficient mantle cell lymphoma cell lines and fresh CLL lymphocytes were also acutely sensitive to vinblastine, a microtubule disrupting drug. Apoptosis induced by vinblastine was dose-dependent with the pharmacologically relevant concentrations of 100-200 nM vinblastine killing CLL cells within 6 hours. The normally recognized mechanism of action of vinblastine suggests that cells are only sensitive in mitosis, yet we find that non-dividing CLL cells are acutely sensitive to this drug. Vinblastine-induced death was both caspase- and c-jun N-terminal kinase (JNK)- dependent, with pan-caspase inhibitor zVAD-fmk and JNK Inhibitor VIII both protecting from vinblastine. In contrast, acute apoptosis induced by ABT-737 is caspase-dependent but JNK-independent. Furthermore, the presence of stromal cells, utilized to mimic the tumor environment of the lymph node, failed to protect CLL cells from ABT-737 or vinblastine. CLL sensitivity to either ABT-737 or vinblastine was independent of CD38 status and previous clinical treatment (untreated or fludarabine, cyclophosphamide and rituximab treated, relapsed patients). These results suggest that vinblastine acts via a JNK-dependent mechanism to kill CLL acutely, while ABT-737 kills acutely via a Bim-dependent mechanism. A number of other leukemia and lymphoma cell lines studied were not acutely sensitive to either ABT-737 or vinblastine. These findings suggest that ABT-737 and vinblastine, alone or in combination, may be beneficial to patients with refractory or minimal residual CLL and mantle cell lymphoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2993.

Role of NOXA and its ubiquitination in proteasome inhibitor-induced apoptosis in chronic lymphocytic leukemia cells

Bortezomib has been successfully used in the treatment of multiple myeloma and has been proposed as a potential treatment for chronic lymphocytic leukemia. In this study we investigated the mechanism by which bortezomib induces apoptosis in chronic lymphocytic leukemia cells. Using western blot analysis, we monitored the regulation of BCL2 family members, proteins of the unfolded protein response (endoplasmic reticulum stress response) and activation of caspases in relation to induction of apoptosis (measured by annexin-propidium iodide staining and loss of mitochondrial membrane potential) by bortezomib in chronic lymphocytic leukemia cells. Bortezomib induced apoptosis through activation of the mitochondrial pathway independently of changes associated with endoplasmic reticulum stress. Perturbation of mitochondria was regulated by a rapid and transcription-independent increase of NOXA protein, which preceded release of cytochrome c, HtrA2, Smac and activation of caspase-9 and -3. NOXA had a short half life (approximately 1-2 h) and was ubiquitinated on at least three primary lysine residues, resulting in proteasomal-dependent degradation. Down-regulation of NOXA, using short interfering RNA in chronic lymphocytic leukemia cells, decreased bortezomib-induced apoptosis. Finally bortezomib when combined with seliciclib resulted in a stronger and earlier increase in NOXA protein, caspase-3 cleavage and induction of apoptosis in chronic lymphocytic leukemia cells. These results highlight a critical role for NOXA in bortezomib-induced apoptosis in chronic lymphocytic leukemia cells and suggest that this drug may become more efficient for the treatment of chronic lymphocytic leukemia if combined with other agents able to interfere with the basal levels of MCL1.

Ethacrynic Acid Exhibits Selective Toxicity to Chronic Lymphocytic Leukemia Cells by Inhibition of the Wnt/β-Catenin Pathway

BackgroundAberrant activation of Wnt/β-catenin signaling promotes the development of several cancers. It has been demonstrated that the Wnt signaling pathway is activated in chronic lymphocytic leukemia (CLL) cells, and that uncontrolled Wnt/β-catenin signaling may contribute to the defect in apoptosis that characterizes this malignancy. Thus, the Wnt signaling pathway is an attractive candidate for developing targeted therapies for CLL.Methodology/Principal FindingsThe diuretic agent ethacrynic acid (EA) was identified as a Wnt inhibitor using a cell-based Wnt reporter assay. In vitro assays further confirmed the inhibitory effect of EA on Wnt/β-catenin signaling. Cell viability assays showed that EA selectively induced cell death in primary CLL cells. Exposure of CLL cells to EA decreased the expression of Wnt/β-catenin target genes, including LEF-1, cyclin D1 and fibronectin. Immune co-precipitation experiments demonstrated that EA could directly bind to LEF-1 protein and destabilize the LEF-1/β-catenin complex. N-acetyl-L-cysteine (NAC), which can react with the α, β-unsaturated ketone in EA, but not other anti-oxidants, prevented the drug's inhibition of Wnt/β-catenin activation and its ability to induce apoptosis in CLL cells.Conclusions/SignificanceOur studies indicate that EA selectively suppresses CLL survival due to inhibition of Wnt/β-catenin signaling. Antagonizing Wnt signaling in CLL with EA or related drugs may represent an effective treatment of this disease.