Upregulation Of Noxa Research Articles

Abstract 1030: Arsenic trioxide plus ethacrynic acid-butyl ester is synergistic to induce apoptosis in myeloid leukemia cells through upregulation of NOXA

Abstract Arsenic trioxide (As2O3) induces complete remission in acute promyelocytic leukemia (APL) patients. However, other types of myeloid leukemia patients are less responsive to As2O3 treatment. APL t(15;17) NB4 cells are more sensitive to As2O3 apoptosis induction than other types of myeloid leukemia cells such as HL-60 and K562 cells. Previously we reported that As2O3 induces apoptosis in NB4 cells through a reactive oxygen species (ROS)-mediated pathway which is attenuated by the expression of glutathione-s-transferase π (GSTπ). NB4 cells contain lower levels of GSTπ than other types of myeloid leukemia cells. Therefore, inhibition of GSTπ should enhance As2O3 induction of apoptosis in other types of myeloid leukemia cells. We investigated the combined effect of As2O3 with ethacrynic acid (EA) and a more potent non-diuretic EA analogue ethacrynic acid-butyl ester (EABE), known GSTπ inhibitors, to induce apoptosis in HL-60 and K562 cells. As2O3 at clinical achievable concentrations (1-2 µM) did not induce apoptosis in HL-60 cells nor in K562 cells. EA at a concentration lower than 60 μM did not induce apoptosis in both cell lines either. As2O3 at 2 μM plus EA at 60 μM are synergistic to induce apoptosis in both HL-60 and K562 cells as measured by Annexin V staining, PARP cleavage and caspase activation. More intriguing, EABE at 1 μM (60 fold lower than EA), which inhibits GSTπ activity, exhibited synergistic induction with As2O3 in K562 cells. Combined index of As2O3 with EABE to induce apoptosis in K562 cells calculated by CompuSyn software was less than 1.0 which indicated synergism. Apoptosis induction by the combination treatment of As2O3 with EABE correlated with the upregulation of the Bcl-2 homology 3 (BH3)-only pro-apoptotic protein NOXA. Knock-down of NOXA using siRNA blocked apoptosis induction of As2O3 plus EABE, while, knock-down of Mcl-1 enhanced the apoptosis induction of As2O3 plus EABE. Immunoprecipiation with Mcl-1 antibody revealed that NOXA bond to Mcl-1 in the K562 cells treated with As2O3 plus EABE. Inhibition of ROS production did not inhibit the upregulation of NOXA nor the apoptosis induction of As2O3 plus EABE treatment. Our data suggest that As2O3 plus EABE induce apoptosis through a novel mechanism which induces NOXA and sequesters Mcl-1 leading to the activation the intrinsic apoptotic pathway. 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 1030.

Abstract 1511: mda-7/IL-24 potently induces apoptosis in human leukemia cells through a process involving induction of ER stress

Abstract mda-7/IL-24, a member of the IL-10 cytokine gene family, has been shown to induce cell death in various epithelial cancer cell types while exhibiting relatively little toxicity toward normal cells. Currently, the effects of mda7/IL-24 on myeloid leukemia cells (AML) have not been extensively characterized. In the present study, we report that treatment with recombinant GST-mda7/IL24 strikingly induces apoptosis in diverse myeloid leukemia cell lines including U937, MV4-11, EOL-1 (both FLT3 mutant), and MLL/ENL cells, which display certain leukemia stem cell-like properties. In addition, mda7/IL24 potently induces apoptosis in and suppresses the colony formation capacity (L-CFU) of primary blasts isolated from multiple patients with AML. In contrast, mda-7/IL-24 exerted only minimal effects on normal CD34+ progenitor cells. These effects were associated with profound mitochondrial injury manifested by cytochrome c and AIF release into the cytosol, caspase-3 activation, and PARP cleavage. The apoptotic effects of mda7/IL24 were associated with a potent induction of ER stress in leukemia cell lines as well as in primary AML blasts, manifested by accumulation of GADD153/CHOP, GADD34, GRP78, and IRE1α. Notably molecular studies with shRNA directed against IRE1α revealed that knockdown of IRE1α significantly enhanced mda7/IL24-mediated apoptosis in U937 cells, suggesting that induction of IRE1α plays a protective role against mda7/IL24 lethality. In addition treatment with mda7/IL24 resulted in down-regulation of the anti-apoptotic protein Mcl-1, and marked accumulation of the pro-apoptotic proteins Bim and Noxa. Notably, ectopic expression of Mcl-1 or knock-down of Bim or Noxa with shRNA markedly decreased mda7/IL24-mediated cell death in leukemia cells. Collectively, these findings indicate that mda7/IL24 strikingly induces apoptosis in human myeloid leukemia through a process that involves ER stress induction, Mcl-1 downregulation, and Bim and Noxa upregulation. They also suggest that mda7/IL24 warrants attention as a potential addition to the therapeutic armamentarium in leukemia and possibly other hematologic malignancies. 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 1511.

Abstract 667: Mechanisms underlying synergistic interactions between the CDK inhibitor flavopiridol (Alvocidib) and the BH3 mimetic GX15-070 (Obatoclax) in human multiple myeloma cells

Abstract To establish a mechanistic basis for a strategy combining CDK inhibitors and Bcl-2 antagonists, in vitro and in vivo interactions between flavopiridol (FP) and GX15-070 (GX) were investigated in human multiple myeloma (MM) cells. The activity of this regimen was also examined in primary CD138+ MM cells and in a murine MM xenograft model. Co-administration of sub-toxic concentrations of FP interacted synergistically with GX to induce apoptosis in U266, RPMI8226, H929, OPM-2, and MM.1S, as well as IL-6-dependent ANBL6 and KAS 6/1 MM cells. Combination treatment also effectively killed MM cells in the presence of stromal cells or growth factors. The regimen was active against multiple drug-resistant MM cell lines, including bortezomib/Velcade- (U266/VR and OPM-2/VR), Revlimid- (8226/R10R), dexamethasone- (MM.1R), melphalan- (8226/LR5), and doxorubicin-resistant (8226/Dox40) MM cells. FP also sharply promoted GX lethality in primary CD138+ MM cells, while sparing normal CD138- bone marrow cells. GX induced clear declines in Mcl-1 and Bcl-xL levels which were potentiated by FP, whereas Bcl-2 expression did not change. Significantly, FP/GX overcame cytoprotective effects mediated by ectopic Mcl-1, Bcl-xL, or Bcl-2 expression. Notably, BH3-only protein expression profiling revealed multiple changes: a) FP, in the presence or absence of GX, triggered a clear increase in Bim expression (both BimEL and BimL isoforms); b) FP/GX co-treatment sharply induced Bik (∼35kDa species) and Noxa (∼27 kDa species),; c) co-exposure (24hr) to FP and GX induced a clear reduction in Puma expression; and c) no changes in the expression of other BH3-only proteins occurred. These findings were confirmed in various MM cell lines, including multiple drug-resistant cells, and were associated with Bak/Bax activation, Bax translocation, cytochrome C release, and caspase activation. Importantly, Bim, Bik, or Noxa knock-down by stable transfection with shRNA dramatically diminished FP/GX lethality. Lastly, co-administration of FP and GX significantly suppressed tumor growth, reflected by diminished tumor size and bioluminescence intensity, in athymic nude mice inoculated with 8226 cells stably expressing luciferase. These events were associated with pronounced apoptosis, manifested by caspase 3 activation and PARP degradation. Western blot analysis also revealed down-regulation of Mcl-1, as well as up-regulation of Bik and Noxa in tumor tissues following FP/GX co-administration. Collectively, these findings provide a mechanistic framework for a strategy combining CDK inhibitors with Bcl-2 antagonists in which down-regulation of anti-apoptotic proteins and induction of BH3-only proteins cooperate to trigger synergistic killing of MM cells, including those resistant to either conventional or novel anti-MM agents, both in vitro and in vivo. 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 667.

Regulation of Endoplasmic Reticulum Stress-induced Cell Death by ATF4 in Neuroectodermal Tumor Cells

The neuroectodermal tumors neuroblastoma and melanoma represent biologically aggressive and chemoresistant cancers. The chemotherapeutic agents fenretinide and bortezomib induce apoptosis through endoplasmic reticulum (ER) stress in these tumor types. The aim of this study was to test the hypothesis that the early events of ER stress signaling and response pathways induced by fenretinide and bortezomib are mediated by the eukaryotic initiation factor 2alpha (eIF2alpha)-ATF4 signaling pathway. Treatment of neuroblastoma and melanoma cell lines with fenretinide, bortezomib, or thapsigargin resulted in induction of eIF2alpha signaling, characterized by increased expression of phosphorylated eIF2alpha, ATF4, ATF3, and GADD34. These events correlated with induction of the pro-apoptotic protein Noxa. The cytotoxic response, characterized by up-regulation of Noxa and cell death, was dependent on ATF4, but not the ER-related pro-death signaling pathways involving GADD153 or IRE1. Although PERK-dependent phosphorylation of eIF2alpha enhanced ATF4 protein levels during ER stress, cell death in response to fenretinide, bortezomib, or thapsigargin was not abrogated by inhibition of eIF2alpha phosphorylation through PERK knockdown or overexpression of wild-type eIF2alpha. Furthermore, ATF4 induction in response to ER stress was dependent primarily on transcriptional activation, which occurred in a PERK- and phosphorylated eIF2alpha-independent manner. These results demonstrate that ATF4 mediates ER stress-induced cell death of neuroectodermal tumor cells in response to fenretinide or bortezomib. Understanding the complex regulation of cell death pathways in response to ER stress-inducing drugs has the potential to reveal novel therapeutic targets, thus allowing the development of improved treatment strategies to overcome chemoresistance.

Abstract B31: Multicellular resistance to bortezomib acquired by mesothelioma spheroids can be reversed by modulation of the Bcl-2 repertoire

Abstract Whereas cancer cells may attain a chemoresistant phenotype on a single cell basis (unicellular resistance), they acquire further resistance to apoptosis when they form three-dimensional (3D) structures (multicellular resistance). The multicellular spheroid, a type of in vitro 3D cell culture, has emerged as a valuable platform to study the acquired multicellular resistance of cancer. We have previously shown that mesothelioma, a recalcitrant tumor with significant apoptotic resistance, manifests a high degree of multicellular resistance in vitro when grown as 3D spheroids. One of the treatments considered for mesothelioma is bortezomib, a proteasome inhibitor; bortezomib has been effective as a single agent in multiple myeloma but has been generally ineffective in solid tumors. Bortezomib has been shown to modulate several pro-/anti-apoptotic members of the Bcl-2 family, including upregulation of pro-apoptotic Noxa, thereby shifting the balance towards apoptosis. We asked whether mesothelioma spheroids develop multicellular resistance to bortezomib and whether changes in the Bcl-2 repertoire could account for the acquired resistance. Using monolayers and spheroids grown from two human mesothelioma cell lines, M28 and REN, we found that these cell lines acquired significant multicellular apoptotic resistance to bortezomib when grown as spheroids. Nonetheless, bortezomib inhibited proteasomal activity equally in both monolayers and spheroids and caused the accumulation of a comparable amount of ubiquitinated proteins in both systems. Despite these similarities, we found that the Bcl-2 repertoire was different in spheroids compared to monolayers, both at baseline and after bortezomib. One major difference was that Noxa, although upregulated in monolayers by bortezomib as expected, was not expressed in the spheroids, either at baseline or after bortezomib. The lack of Noxa accounted for some of the resistance because we found that a cell-permeable Noxa-BH3 peptide partially restored the apoptotic response to bortezomib in 3D spheroids. ABT-737, an inhibitor of the anti-apoptotic proteins Bcl-3/xL/w, was even more effective at reversing bortezomib resistance acquired by cells in 3D. In fact, when given alone, ABT-737 was selectively toxic for the spheroids, inducing apoptosis by itself in 3D spheroids but not in the 2D monolayers. In conclusion, we find that Bcl-2 proteins can account for apoptotic resistance in 3D. Manipulation of the Bcl-2 repertoire may be effective alone and may also improve the therapeutic response of mesothelioma to bortezomib therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B31.

Displacement of Bim From Anti-Apoptotic Proteins Is the Primary Factor for Determining ABT-737 Activity in Multiple Myeloma Cell Lines.

Abstract 2851Poster Board II-827ABT-737 and its orally active analog ABT-263 are Bcl-2-family inhibitors that are currently in clinical trials for a variety of cancers including hematological malignancies such as multiple myeloma. Previously, we reported that the sensitivity of multiple myeloma cell lines to ABT-737 correlates with the interactions, but not the expression, of Bcl-2 proteins. Analysis of 6 multiple myeloma cell lines revealed that expression of Bcl-2 proteins did not correlate with sensitivity, however the sensitive cells (8226/S, MM.1S and KMS-11) have a substantial amount of their pro-apoptotic Bcl-2 protein, Bak, bound to Bcl-xL. On the other hand, in the insensitive cell lines (U266, KMS-11 and OPM2), Bak was found to be associated with Mcl-1, a family member that does not bind ABT-737 and thereby confers resistance to this drug. Furthermore, we also showed that release of the BH3-only protein Bim by ABT-737 from Bcl-xL and Bcl-2 also contributes to cell death in 8226/S and MM.1S. The purpose of the current study is to further investigate the role of Bim in ABT-737-induced cell death in the multiple myeloma lines. Similar to Bak, a substantial amount of Bim is bound to Bcl-xL and Bcl-2 in the ABT-737-sensitive cell lines, MM.1S and KMS-18, while in the insensitive cell lines, it is highly bound to Mcl-1. Surprisingly, in the ABT-737-sensitive 8226/S cells, Bim appears to bind to Mcl-1. However in these cells, ABT-737 treatment resulted in upregulation of Noxa, which is a BH3-only protein that binds Mcl-1 and can release Bim. Taken together these data suggest that although binding of Bim to Mcl-1 may confer resistance to ABT-737, in certain cell types this treatment could also induce Noxa expression that antagonizes Mcl-1-mediated resistance. Consistent with this hypothesis, Mcl-1 overexpression as well as knockdown of Noxa expression significantly protected 8226/S cells from ABT-737-induced cell death while they had no effect in MM.1S cells. To further demonstrate the role of Bim in ABT-737-induced cell death, ABT-resistant 8226/S, KMS-11, KMS-18 and U266 cell lines were generated. In the resistant cell lines of 8226/S and KMS-18, Bim is exclusively bound to Mcl-1, which was overexpressed as compared to the parental cells. Bak binding was not affected by acquisition of ABT-737 resistance. This result is in agreement with the findings that interaction of Bim and Mcl-1 confers resistance to ABT-737. On the other hand, in ABT-resistant U266 and KMS-11 cell lines, Bim expression was down-regulated while Mcl-1 levels were not changed. Thus, it appears that in cells where Bim is already bound to Mcl-1, further resistance is achieved by down-regulating the expression of this BH3-only protein. Overall, these results suggest that the complex interactions between Bcl-2 proteins need to be investigated in order to understand how multiple myeloma cells may respond to ABT-737 treatment. Disclosures:Boise:University of Chicago: Patents & Royalties.

Targeting CDK9 Dramatically Potentiates ABT-737-Induced Apoptosis in Human Multiple Myeloma Cells through a Bim-Dependent Mechanism.

Abstract 297 Background:High expression of the anti-apoptotic Bcl-2 family protein Mcl-1 is a critical survival mechanism in human multiple myeloma (MM) cells, and a key barrier to apoptosis induced by the Bcl-2/-xL antagonist ABT-737. De novo expression of the short-lived protein Mcl-1 is controlled transcriptionally by CDK9, a catalytic subunit of the PTEF-b transcription elongation complex (CDK9/cyclin T) that phosphorylates the C-terminal domain (CTD) of RNA polymerase II (RNA pol II) to promote elongation of nascent transcripts. Thus, approaches targeting CDK9 may enhance ABT-737 efficacy in MM cells. Methods:To test this hypothesis, we employed the pan-CDK inhibitor roscovitine and a highly-selective CDK9 inhibitor (CDK9i) in drug-naïve and drug-resistant MM cell lines, as well primary plasma cells. Results:Co-administration of non- or sub-toxic concentrations of roscovitine or CDK9i interacted in a highly synergistic manner with ABT-737 (e.g., 150-300 nM) to induce apoptosis in U266, RPMI 8226, H929, OPM-2, and MM.1S MM cells, as well as IL-6-depedent ANBL-6 and KAS 6/1 cells. In each case, Combination Index values were significantly less than 1.0. These regimens were fully active in various drug-resistant MM cells, including U266/VR and OPM-2/VR bortezomib-resistant cells, RPMI 8226/RR lenalidomide-resistant cells, MM.1R dexamethasone-resistant cells, RPMI 8226/LR5 melphalan-resistant cells, and RPMI 8226/Dox40 doxorubicin-resistant cells. They also effectively killed MM cells in the presence of stromal cells or growth factors (i.e., IL-6, IGF-1, BAFF, and APRIL). Co-administration of roscovitine significantly enhanced ABT-737 lethality in primary CD138+ MM cells, while largely sparing CD138− bone marrow cells. Notably, the majority of MM cell lines exhibited high expression of cyclin T1 rather than cyclin T2a/b, in association with constitutively-activated CDK9, manifested by high levels of phosphorylation of CDK9 and RNA pol II CTD at Ser2 and 5. Exposure of MM cells to either roscovitine or CDK9i strikingly reduced RNA pol II CTD phosphorylation at both sites (particularly Ser5), but did not affect CDK9 and cyclin T1 levels. Whereas treatment with ABT-737 alone led to a modest but clear increase in Mcl-1 expression, roscovitine or CDK9i completely blocked this event and substantially down-regulated basal Mcl-1 expression. Notably, similar phenomena occurred in U266/VR cells, which displayed higher basal levels of Mcl-1, an important candidate resistance mechanism, compared to parental cells. Moreover, knock down of Mcl-1 by stable transfection with shRNA dramatically increased U266 sensitivity to ABT-737. Co-treatment of MM cells with ABT-737 and roscovitine or CDK9i did not affect expression of other anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL, but induced a Bcl-2 cleavage fragment. Expression profiling of BH3-only proteins in MM cells co-exposed to roscovitine and ABT-737 revealed a modest or a marked increase in expression of Bim (both EL and L isoforms) and Noxa (particularly a ∼27 kDa species), respectively, accompanied by a clear decrease in Puma levels. No changes in expression of other BH-3-only proteins (e.g., Bid, Bad, Bik, BMF, Hrk) were observed. Interestingly, knock down of Bim by stable transfection with shRNA dramatically diminished roscovitine/ABT-737 lethality, whereas Noxa knock-down by shRNA only partially protected cells from this regimen. In contrast, both shRNA approaches substantially protected U266 cells from bortezomib lethality. Conclusions:Collectively, these findings provide a mechanistic framework for targeting transcription by CDK9 inhibitors to diminish ABT-737-mediated Mcl-1 upregulation and to downregulate basal Mcl-1 levels, resulting in highly synergistic killing of MM cells, including those resistant to either conventional or novel anti-MM agents. They also suggest that the BH3-only protein Bim may be required for apoptosis triggered by this strategy, while up-regulation of Noxa may amplify MM cell killing in this setting. Disclosures:No relevant conflicts of interest to declare.

ROS-Mediated Upregulation of Noxa Overcomes Drug-Resistance Due to P53-Dysfunction in Chronic Lymphocytic Leukemia.

Abstract 2382Poster Board II-359Although recent advances in treatment-strategies for chronic lymphocytic leukemia (CLL) have resulted in increased remission rates and response duration, the disease eventually relapses, which necessitates repeated cycles of therapy. Eventually most patients develop chemo-resistant disease which infers a very poor prognosis. The activity of purine-analogs and alkylating agents, the backbone of current treatment regimens, depends on functional p53 and chemo-resistance is highly associated with a dysfunctional p53-response.P53-independent sensitization of CLL cells to these compounds could represent a novel strategy to overcome chemo-resistance. Platinum-based compounds have been successfully applied in relapsed lymphoma and recently also in high-risk CLL. In various cancer-types, the activity of such compounds has been found to be p53-independent and in part mediated by p73. In this study we investigated the efficacy and mechanism of action of platinum-based compounds in chemo-refractory CLL.Neither cisplatinum nor oxaliplatin as a single agent induced cell death in clinically relevant doses. However, independent of p53-functional status, platinum-based compounds acted synergistically with fludarabine, which was found to be caspase-dependent. Combination-treatment resulted in strong upregulation of the pro-apoptotic BH3-only protein Noxa. We did not find evidence for a role of p73; however, the observed synergy was found to involve generation of reactive oxygen species (ROS). Co-treatment with ROS-scavengers completely abrogated Noxa-upregulation and cell-death upon combination treatment in p53-dysfunctional CLL. Noxa RNA-interference markedly decreased sensitivity to combination treatment, supporting a key role for Noxa as mediator between ROS signaling and apoptosis induction.In addition to these findings, we tested the effects of platinum-based compounds and fludarabine on drug-resistance resulting from CD40-ligand stimulation of CLL cells, which represents a model for CLL cells in the protective micro-environment of the secondary lymph node-tissue (Hallaert et al Blood 2008 112(13):5141). Combination treatment could overcome CD40-ligand induced chemo-resistance and was, at least in part, mediated by the generation of ROS and marked induction of expression of Noxa.Our data indicate that interference with the cellular redox-balance represents an interesting target to overcome drug resistance due to both p53-dysfunction as well as micro-environmental protective stimuli in CLL. Disclosures:No relevant conflicts of interest to declare.

Induction of Noxa Via Generation of Reactive Oxygen Species Plays a Key Role in Induction of CLL Cell Apoptosis by Two Novel p53-Independent Cytotoxic Agents.

Abstract 2752Poster Board II-728Approximately 15% of CLL patients present with a deletion of chromosome 17p, resulting in the loss of the p53 gene. The percentage of 17p-deleted patients can increase to between 30–50% following treatment with cytotoxic agents which induce CLL apoptosis via upregulation of p53. Therefore, identification of novel agents which kill CLL cells independently of p53 is of crucial importance. We have recently shown that two chemically unrelated agents, 2-phenylacetylenesulfonamide (PAS) and the sesquiterpene lactone LC1 kill CLL cells regardless of the functional status of p53. Furthermore, in contrast to the conventional drugs chlorambucil and fludarabine, PAS and LC1 induce apoptosis in the absence of p53 elevation. However, the mechanisms of action by which these agents induce p53-independent apoptosis are unclear. We have previously shown that both PAS and LC1 initiated CLL cell apoptosis within 6-10h with, maximal killing by 48h. Here we show that treatment of CLL cells with either agent results in the generation of reactive oxygen species (ROS), the activating phosphorylation of the pro-apoptotic MAP kinase family member JNK (Fig 1), resulting in turn in upregulation of its downstream target, the transcription factor c-JUN (Fig 2). The BH3-only pro-apoptotic protein Noxa was originally described as a pro-apoptotic target for upregulation by p53. However, both PAS (Figs1 and 2) and LC1 upregulated Noxa in a p53-independent manner. Addition of N-acetylcysteine (NAC), a free radical scavenger, decreased ROS generation by PAS or LC1 and also prevented phosphorylation of JNK and Noxa upregulation (Fig 1) and also the upregulation of c-JUN. NAC also strikingly abrogated apoptosis induction by either agent, as shown by quantitation of cleavage of the caspase 3 substrate poly (ADP ribose) polymerase (PARP; Fig 1). Taken together, the data suggest both PAS and LC1 induce p53-independent apoptosis via upregulation of ROS and the subsequent induction of Noxa. The data are also compatible with a role for JNK and c-JUN in the events leading to Noxa upregulation. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The PI3K Inhibitor ON 01910.Na Inhibits Critical Survival Pathways and Induces Apoptosis in CLL Cells through Induction of NOXA and BIM.

Abstract 412Chronic Lymphocytic leukemia (CLL), the most common leukemia in Western countries, is a clinically heterogeneous disease characterized by the accumulation of mature B lymphocytes. Disease progression is driven by cell survival and proliferation signals provided by the tumor microenvironment through different receptors including the B-cell receptor, TNF, chemokine and cytokine receptors, and through other cell-cell interactions. ON 01910.Na (Onconova, Therapeutics) belongs to the family of styryl benzyl sulfones, a novel family of non-ATP competitive compounds that are currently under development as potential anticancer treatments. These compounds have shown activity in a variety of xenograft mouse models. Interestingly, it has recently been described that ON 01910.Na exerts potent antitumor activity against Mantle Cell Lymphoma (MCL) cells by inhibition of PI-3K/Akt/mTOR pathway and downregulation of Cyclin D1 translation. Here, we evaluated the cytotoxic effect of ON 01910.Na after 48 hours of in vitro exposure on PBMCs from CLL and normal donors by flow cytometry using AnnexinV-PE. We co-stained with CD3-APC and CD19-FITC to assess the effect on B and T cells separately. ON 01910.Na induced apoptosis of the leukemic cells in all CLL samples tested (n=24), without affecting T-cell viability. Notably, ON 01910.Na did not affect B and T cells from normal donors at concentrations that induced apoptosis of CLL cells. Thirteen CLL samples were highly sensitive towards ON 01910.Na (mean B-cell IC50 0.71 μM) a concentration readily achieved in phase I clinical trials, eight samples were sensitive (mean B-cell IC50 1.38 μM) and three showed moderate sensitivity (mean B-cell IC50 4.26 μM). In contrast, the IC50 of T-cells was >8 μM in all samples. No significant differences were found in ON 01910.Na cytotoxicity against CLL expressing mutated (n=9, mean B-cell IC50 1.45 μM) or unmutated (n=15, mean B-cell IC50 1.34μM) IgVH sequences. ON 01910.Na was also effective against tumor cells having 17p (n=4, mean B-cell IC50 1.45 μM) or 11q deletions (n=3, mean B-cell IC50 0.96 μM). ON 01910.Na activated the mitochondrial apoptotic pathway, as shown by flow cytometry using conformation specific antibodies to detect activation of Bax and Bak, leading to mitochondrial depolarization (measured by mitotracker) and caspase-3 activation. ON 01910.Na also induced the upregulation of the proapoptotic BH3-only proteins Noxa and Bim and decreased expression of Mcl-1 at 4 hours, well before the onset of apoptosis. Inhibition of Noxa expression by retroviral transfection reduced the sensitivity to drug by 60%. ON 01910.Na induced reactive oxygen species (ROS) that may contribute to Noxa induction. Consistently ROS blockade using N-acetyl-cysteine reduced ON 01910.Na cytotoxic activity. To further delineate the biological processes 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. GEP revealed the induction of Noxa and ATF3, consistent with an oxidative stress response. Noteworthy, Gene Set Enrichment Analysis (GSEA) of microarray data showed an induction of an AP-1 gene signature, (False Discovery Rate (FDR) = 0.10), validated by the nuclear accumulation of c-jun at 4 hours and a FOXO gene signature (FDR=0.13) that correlated with the upregulation of the proapoptotic BH3-only protein Bim. Strikingly, ON 01910.Na repressed B-cell receptor (FDR=0.023), NF-kB (FDR=0.084) and PI3K gene signatures. Consistent with inhibition of the BCR/PI3K/AKT axis, ON 01910.Na inhibited AKT phosphorylation by 65% after in vitro BCR activation of CLL cells. Our results identify ON 01910.Na as a promising agent in the treatment of CLL with an interesting dual mechanism of action: activation of apoptotic stress signals leading to Noxa and BIM up-regulation, combined with inhibition of the BCR/PI3K/AKT pathway that can block microenvironment-induced survival and proliferation signals. These data support the development of ON 01910.Na in CLL and a clinical trial has been initiated at our institution. Disclosures:No relevant conflicts of interest to declare.

NRF2 at the Interface of Oxidative and Endoplasmic Reticulum Stress Pathways Is a Critical Integrator of Bortezomib Response in Mantle Cell Lymphoma in Vitro and In Vivo.

Abstract 727The proteasome inhibitor bortezomib (BZM) is effective as single-agent in relapsed and refractory mantle cell lymphoma (MCL) but more than half of patients remain insensitive to BZM. Suggested mechanisms of action include activation of Noxa, p53, oxidative, and endoplasmic reticulum (ER) stress. To define mechanisms relevant for BZM-induced cytotoxicity we pursued two approaches: first, we characterized gene expression changes in 10 MCL cell lines exposed for 24h to 10nM BZM, a concentration that kills >50% of sensitive, but <20% of resistant cells. Secondly, we analyzed gene expression changes in tumor cells of patients with leukemic MCL undergoing BZM treatment in a clinical trial. RNA was extracted from MCL cell lines at early (1h and 3h), intermediate (6h) and late (24h) time points. Virtually no changes in gene expression were detectable at 1 and 3h of drug exposure and only about 100 genes changed by 6h. After 24h of treatment 524 genes were significantly changed in sensitive and 271 genes in resistant cell lines respectively. The delayed onset of gene expression changes is consistent with the reversibility of BZM toxicity for up to 8 hours. Using Ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA) we identified two dominant responses induced by BZM: 1) an oxidative stress response mediated by NRF2 and related transcription factors, and 2) an ER stress/ubiquitin proteasome response (FDR by GSEA <0.1). Both responses were primarily apparent in sensitive cell lines. A set of 20 experimentally validated NRF2 target genes was used as a core NRF2 signature: this signature was increased 15-fold on average in sensitive cell lines but only 2-fold in resistant cell lines (P=0.006). Similarly, an XBP1 and ATF6 signature, reflecting activation of the ER stress response, was stronger induced in sensitive than in resistant cell lines (average 1.9-fold vs 1.3-fold; P=0.003). Activation of these stress pathways upon BZM treatment was confirmed by demonstrating accumulation of nuclear NRF2 in sensitive Jeko1 but not in resistant Mino cells. Also markers of ER stress such as phosphorylation of ER resident nuclease Ire1 that splices the transcription factor XBP1 and activation of ATF3, ATF4, and CHOP downstream of PERK were readily detected in Jeko1 but not in Mino cells. Finally, Noxa, the BH3-only protein primarily responsible for BZM-induced apoptosis, was only induced in sensitive Jeko1 cells. We next analyzed the effect of BZM on purified tumor cells from five patients with leukemic MCL treated with BZM (1.5mg/m2, day 1, 4, 8 and 11). Two patients showed a >50% reduction in circulating tumor cells after 2 injections of drug (day 8) and >75% reduction after 4 injections (day 2, sensitive), while in three patients there was no change or an intermediate response (resistant). Western blotting demonstrated Noxa up-regulation in circulating tumor cells of sensitive but not resistant samples. This is consistent with the demonstrated importance of Noxa for induction of apoptosis in response to BZM in cell line studies. Next we performed gene expression profiling immediately before, at 6h, and 24h after the first and 24h after the second dose of BZM. Using IPA and GSEA up-regulation of the ubiquitin/proteasome pathway and the NRF2-mediated oxidative stress response was again prominent, but an ER stress response was less apparent. XBP1 splicing was not detected in tumor cells from sensitive samples indicating that an ER stress response was not fully activated by BZM in vivo. Consistent with in vitro data the NRF2 signature was induced 2.3-fold on average in sensitive but not in resistant samples (P<0.05). Intriguingly, baseline expression of the NRF2 signature genes was significantly higher in resistant than in sensitive cells (P=0.0007). In summary, we identify NRF2 as critical integrator of different stress pathways in response to BZM in MCL. Thus, rapid induction of NRF2 target genes might be a useful biomarker of BZM-induced cellular stress and predict clinical response. Our data suggest a more complex function of NRF2 than previously appreciated. NRF2-regulated genes serve primarily homeostatic roles and enable cells to deal with oxidative and xenobiotic insults; a function that may come to play in BZM resistant cells with higher baseline expression of NRF2 target genes. On the other hand, our data suggest a possible pro-apoptotic role of acute induction of high levels of NRF2 that is currently under investigation. Disclosures:No relevant conflicts of interest to declare.

Cytoplasmic p53 and Activated Bax Regulate p53-dependent, Transcription-independent Neural Precursor Cell Apoptosis

The prodeath effects of p53 are typically mediated via its transcriptional upregulation of proapoptotic Bcl-2 family members, including PUMA, Noxa, and/or Bax. We previously reported that staurosporine (STS), a broad-spectrum kinase inhibitor and prototypical apoptosis-inducing agent, produced p53-dependent, Bax-dependent, neural precursor cell (NPC) apoptosis, but that this effect occurred independently of new gene transcription and PUMA expression. To further characterize the mechanism by which p53 regulates NPC death, we used primary cerebellar NPCs derived from wild-type, p53-deficient, and Bax-deficient neonatal mice and the mouse cerebellar neural stem cell line, C17.2. We found that STS rapidly increased p53 cytoplasmic immunoreactivity in neuritic-like processes in C17.2 cells, which preceded Bax activation and caspase-3 cleavage. Confocal microscopy analysis of STS-treated cells revealed partial colocalization of p53 with the mitochondrial marker pyruvate dehydrogenase as well as with conformationally altered "activated" Bax, suggesting an interaction between these proapoptotic molecules in triggering apoptotic death. Nucleophosmin (NPM), a CRM1-dependent nuclear chaperone, also exhibited partial colocalization with both activated Bax and p53 following STS treatment. These observations suggest that cytoplasmic p53 can trigger transcription-independent NPC apoptosis through its potential interaction with NPM and activated Bax.

Human Melanoma Cells under Endoplasmic Reticulum Stress Are More Susceptible to Apoptosis Induced by the BH3 Mimetic Obatoclax

Past studies have shown that melanoma cells have largely adapted to endoplasmic reticulum (ER) stress, and this is associated with up-regulation of the antiapoptotic proteins Bcl-2 and Mcl-1. In this report, we show that the BH3 mimetic obatoclax potently overcomes resistance of melanoma cells to apoptosis induced by ER stress. Obatoclax, as a single agent at nanomolar concentrations, was relatively ineffective in the induction of apoptosis in melanoma cells, but treatment with obatoclax at these concentrations in combination with the ER stress inducer tunicamycin (TM) or thapsigargin markedly enhanced apoptotic cell death. This was primarily because of the inhibition of Mcl-1 by obatoclax, in that cotreatment with TM and another BH3 mimetic ABT737, which does not antagonize Mcl-1, caused only minimal increases in apoptosis. Moreover, overexpression of Mcl-1 inhibited apoptosis to greater degrees than overexpression of Bcl-2. In addition to direct inhibition of Mcl-1 by obatoclax, the combination of obatoclax and TM caused strong up-regulation of the BH3-only protein Noxa. Small RNA interference knockdown of Noxa partially inhibited apoptosis induced by cotreatment with obatoclax and TM. Similarly, knockdown of Bak also blocked induction of apoptosis by the compounds. The Mcl-1/Bak interaction seemed to be disrupted more efficiently in melanoma cells cotreated with obatoclax and TM. Taken together, these results identify obatoclax as a potent agent that overcomes resistance of melanoma cells to ER stress-induced apoptosis and seem to have important implications in the use of BH3 mimetics in the treatment of melanoma.

Apoptotic Signaling Activated by Modulation of the F0F1-ATPase: Implications for Selective Killing of Autoimmune Lymphocytes

7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(napthalen-2-ylmetyl)-4,5,-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423) is a proapoptotic 1,4-benzodiazepine that potently suppresses disease in the murine model of lupus by selectively killing pathogenic lymphocytes. In MRL/MpJ-Fas(lpr) (MRL-lpr) mice, Bz-423 overcomes deficient expression of the Fas death receptor and hyperactivation of antiapoptotic phosphatidylinositol 3-kinase (PI3K)-Akt signaling to specifically kill pathogenic CD4(+) T cells. Bz-423 binds to the oligomycin-sensitivity-conferring protein component of the mitochondrial F(0)F(1)-ATPase, which modulates the enzyme leading to formation of superoxide by the mitochondrial respiratory chain. Scavenging this reactive oxygen species blocks all subsequent components of the apoptotic cascade. To gain insight into how apoptotic signaling activated by Bz-423-induced superoxide contributes to the selective depletion of MRL-lpr CD4(+) T cells, we characterized the death mechanism in a CD4(+) T cell leukemia line (Jurkat). Although Bz-423-induced superoxide indirectly inactivates Akt, this response is not required for T cell death. Apoptosis instead results from parallel increases in levels of the proapoptotic Bcl-2 proteins Noxa and Bak leading to specific activation of Bak, mitochondrial outer membrane permeabilization, and a commitment to apoptosis. By directly up-regulating proteins that trigger loss of mitochondrial outer membrane integrity, Bz-423 bypasses defective Fas function and antiapoptotic PI3K-Akt signaling in MRL-lpr CD4(+) T cells. Moreover, because disease-associated abnormalities should sensitize autoreactive CD4(+) T cells to transcriptional up-regulation of Noxa by redox signals and to Bak-dependent apoptosis, the apoptotic mechanism elucidated in Jurkat cells provides important clues into the cell-type- and disease-selective effects of Bz-423 in MRL-lpr mice.

BAX/BAK–Independent Mitoptosis during Cell Death Induced by Proteasome Inhibition?

Proteasome inhibitors induce rapid death of cancer cells. We show that in epithelial cancer cells, such death is associated with dramatic and simultaneous up-regulation of several BH3-only proteins, including BIK, BIM, MCL-1S, NOXA, and PUMA, as well as p53. Elevated levels of these proteins seem to be the result of direct inhibition of their proteasomal degradation, induction of transcription, and active translation. Subsequent cell death is independent of BAX, and probably BAK, and proceeds through the intrinsic mitochondrial apoptosis pathway. We identify the cascade of molecular events responsible for cell death induced by a prototypical proteasome inhibitor, MG132, starting with rapid accumulation of BH3-only proteins in the mitochondria, proceeding through mitochondrial membrane permeabilization and subsequent loss of DeltaPsi(m), and leading to irreversible changes of mitochondrial ultrastructure, degradation of mitochondrial network, and detrimental impairment of crucial mitochondrial functions. Our results also establish a rationale for the broader use of proteasome inhibitors to kill apoptosis-resistant tumor cells that lack functional BAX/BAK proteins.

2-phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells

AbstractWe studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 μM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.

HDAC2 mediates therapeutic resistance of pancreatic cancer cells via the BH3-only protein NOXA

Background:Although histone deacetylase inhibitors (HDACi) are promising cancer therapeutics regulating proliferation, differentiation and apoptosis, molecular pathways engaged by specific HDAC isoenzymes in cancer are ill defined.Results:In this study we demonstrate...

Effect of (-)-gossypol (AT-101) on transcriptional regulation of Noxa and Puma and on Mcl-1-mediated cancer cell resistance to apoptosis

e14611 Background: AT-101 is being evaluated in Phase II clinical trials for the treatment of human cancers and demonstrates anticancer activity as a single agent and in combination with docetaxel. Despite extensive investigations, the mechanism of action for its antitumor activity is not completely understood. In this study, we have performed detailed investigations using a panel of human cancer cell lines on the mechanism of action for AT-101. Methods: A series of human breast and prostate cancer cell lines were utilized to evaluate the antitumor activity of AT-101. Cell growth inhibition, cell viability and apoptosis were determined by WST assay, trypan blue assay and flow cytometry, respectively. Gene specific siRNAs were used to knockdown target gene expression. Quantitative RT- PCR was performed to detect the mRNA levels of Puma and Noxa. Co-immunoprecipitation was carried out to detect the association between Mcl-1 and Noxa or Puma. Results: AT-101 induced apoptosis in both Bax/Bak-dependent and -independent manners in a variety of human cancer cell lines concomitant with increased expression of Noxa and Puma in a p53-independent manner. The up-regulation of Noxa was not observed in normal primary breast epithelial cells or normal-like human breast epithelial MCF-12F cells. Transcription inhibitor actinomycin D blocked AT-101-induced up-regulation of Puma and Noxa. Knockdown of Noxa or Puma attenuated AT-101-induced apoptosis. Furthermore, AT-101 effectively overcame Mcl-1-mediated cancer cell resistance to apoptosis. Co-immunoprecipitation studies demonstrated that Mcl-1 specifically associated with Noxa but not Puma. Conclusions: Our findings suggest that transcriptional up-regulation of pro-apoptotic Noxa and Puma contributes to the antitumor activity of AT-101, which plays a dominant role in antagonizing Mcl-1 and overcoming Mcl-1-mediated resistance to apoptosis of cancer cells. AT-101 functions as an antagonist of Bcl-2, Bcl-xL and Mcl-1 through direct binding to these proteins, as well as through upregulation of Noxa and Puma. [Table: see text]

A phase I study of R-(-)-gossypol (AT-101) in combination with cisplatin (P) and etoposide (E) in patients with advanced solid tumors and extensive-stage small cell lung cancer (ES-SCLC)

e13502 Background: AT-101 [R-(-)-gossypol acetic acid] (AT) is an orally-administered BH3 mimetic that lowers the threshold for apoptosis by direct binding to Bcl-2, Bcl-xL, Mc1–1, Bcl-W, and through upregulation of the proapoptotic proteins Noxa and Puma. Bcl- 2 is over-expressed in &gt;80% of SCLC. In vitro study using SCLC cells showed that treatment with EP had synergistic cytotoxic effects in suppression of Bcl-2. This is a phase I dose-escalation study of AT in combination with EP with an expanded cohort of patients with ES-SCLC. This study is being conducted to determine the maximum tolerated dose (MTD), pharmacokinetics and activity of AT with EP ± pegfilgrastim (F) in patients with advanced, refractory solid tumors and/or ES-SCLC. Methods: This study used standard eligibility criteria except patients must not have received prior therapy that inhibits the Bcl-2 family. At dose level 1, patients received P 60 mg/m2 on day 1 and E 100 mg/m2 on days 1, 2, and 3 every 21 days. AT was administered 30 mg orally BID on days 1, 2 and 3 of each cycle. Results: 10 patients have been enrolled; 7 men, 3 women. Tumor types: 6 lung; 2 prostate; 1 head &amp; neck; 1 unknown primary. 2 of 5 patients enrolled at dose level 1 experienced a DLT of neutropenic fever in cycle 1. Three subsequent patients were enrolled to dose level -1 (20 mg BID x 3 days, d1–3) which was well tolerated. Additional patients are being enrolled at dose level 1a (EP+AT with F). Grade 3/4 toxicities related to AT without F at dose level 1 and -1 were as follows: ANC (8), leucopenia (7), febrile neutropenia (2), low hemoglobin (1), thrombocytopenia (1), elevated AST (1), cardiac ischemia/MI (1), diarrhea (1). There were no reported grade 3/4 toxicities in two patients at level 1a. Four patients had stable disease; two progressive disease and four patients were unevaluable. Conclusions: The MTD without F was established at AT 20mg orally BID, P 60 mg/m2 on day 1, and E 100 mg/m2 on days 1, 2, and 3 every 21 days. The MTD with F has not yet been established. Accrual continues at dose level 1a. Subjective and objective evidence of clinical activity has been observed in patients with refractory solid tumors. This study was supported by NCI, UO1 CA062491, SAIC 25XS097 and 1ULRR025011. No significant financial relationships to disclose.

ABT-737 Synergizes with Chemotherapy to Kill Head and Neck Squamous Cell Carcinoma Cells via a Noxa-Mediated Pathway

Overexpression of Bcl-X(L), an antiapoptotic Bcl-2 family member, occurs in a majority of head and neck squamous cell carcinomas (HNSCCs) and correlates with chemotherapy resistance in this disease. Overexpression of Bcl-2 is also observed in HNSCC, albeit less frequently. We have previously shown that peptides derived from the BH3 domains of proapoptotic proteins can be used to target Bcl-X(L) and Bcl-2 in HNSCC cells, promoting apoptosis. In this report, we examined the impact of ABT-737 (for structure, see Nature 435: 677-681, 2005 ), a potent small-molecule inhibitor of Bcl-X(L) and Bcl-2, on HNSCC cells. As a single agent, ABT-737 was largely ineffective at promoting HNSCC cell death. By contrast, ABT-737 strongly synergized with the chemotherapy drugs cisplatin and etoposide to promote HNSCC cell death and loss of clonogenic survival. Synergism between ABT-737 and chemotherapy was associated with synergistic activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. Treatment with ABT-737 plus chemotherapy resulted in dramatic up-regulation of proapoptotic Noxa protein, and small interfering RNA (siRNA)-mediated inhibition of Noxa up-regulation partially attenuated cell death by the synergistic combination. Treatment with cisplatin or etoposide, alone or in combination with ABT-737, resulted in substantial down-regulation of Mcl-1L, a known inhibitor of ABT-737 action. Further down-regulation of Mcl-1L using siRNA failed to enhance killing by the cisplatin/ABT-737 synergistic combination, indicating that chemotherapy treatment of HNSCC cells is sufficient to remove this impediment to ABT-737. Together, our results demonstrate potent synergy between ABT-737 and chemotherapy drugs in the killing of HNSCC cells and reveal an important role for Noxa in mediating synergism by these agents.