ALBERT

Description: Abstract 3415 The advent of imatinib, a Bcr-Abl tyrosine kinase inhibitor (TKI) has revolutionized the treatment of patients with CML. Development of resistance and limited activity in blast crisis (BC) CML are evolving problems facing this therapy. We found that XIAP, a potent caspase inhibitor, is highly expressed in CML cells, in both, cell lines and patient samples. Treatment with imatinib deceased XIAP levels in imatinib-sensitive KBM5 but much less so in imatinib-resistant KBM5STI571 cells (harboring T315I mutation) suggesting that XIAP expression in CML is regulated at least in part via Bcr-Abl and that targeting XIAP may promote cell death in CML cells by circumventing imatinib resistance. To test this, we treated BC CML cells with XIAP antisense oligonucleotide (ASO) and with SMAC mimetic ABT-10 and found that inhibition of XIAP induced apoptotic cell death with similar efficacy in KBM5 cells and KBM5STI517 cells (EC50=6.3±0.3 μM and 8.4±0.4 μM at 48 hours, respectively for ABT-10). However, we noted that inhibition of XIAP by ASO induced the expression, in both KBM5 and KBM5STI571 cells, of apoptosis repressor with caspase recruitment domain (ARC) in both mRNA and protein levels but not the expression of Bcl-2 protein. ARC is a unique antiapoptotic protein. It acts through inhibiting caspases and antagonizing the activity and function of p53 and Bax. Therefore, its induction may antagonize the effect of XIAP downregulation. Indeed, inhibition of both XIAP and ARC by ASO induced significantly more cell death than inhibiting either protein alone in both KBM5 and KBM5STI cells. Furthermore, we demonstrated that XIAP inhibition induced-apoptosis was enhanced by imatinib in KBM5, but not in KBM5STI cells. Interestingly, inhibition of Bcr-Abl tyrosine kinase by imatinib not only decreased XIAP, but also suppressed ARC levels in KBM5 but had minimal effects on the levels of these proteins in KBM5STI571 cells and enforced expression of the Bcr-Abl p185 fusion protein (in HL-60 cells) greatly increased both XIAP and ARC levels. This induction was inhibited by imatinib suggesting that ARC is also a downstream target of Bcr-Abl tyrosine kinase. Therefore, imatinib enhancing XIAP inhibition induced-apoptosis in KBM5, not KBM5STI cells can be explained at least in part by its ability to decrease XIAP and ARC levels. In conclusion, XIAP is highly expressed in CML cells and upregulated by Bcr-Abl. Targeting XIAP promotes death of BC and TKI resistant CML cells. Results suggest that XIAP is a potential target in BC and TKI resistant CML cells and that XIAP inhibition-induced apoptosis is enhanced by imatinib in TKI sensitive cells and by ARC inhibition independent of cellular responses to TKIs. Inhibition of XIAP and ARC as a novel therapeutic strategy in CML warrants further investigation. Disclosures: Koller: Isis Pharmaceuticals: Employment.

Description: TR3 (Nur77) is an orphan member of the steroid/retinoid family of Nuclear Receptors (NRs) that translocates from nucleus to cytosol, where it binds Bcl-2 and converts its phenotype from anti-apoptotic to pro-apoptotic (Li, et al. Science289: 1159, 2000; Lin, et al CELL116: 527, 2004). We surveyed TR3 (Nur77) for interactions with all six human anti-apoptotic Bcl-2-family proteins: Bcl-2, Bcl-XL, Mcl-1, Bcl-W, Bfl-1, and Bcl-B. We observed that Bcl-2, Bfl-1, and Bcl-B interact with TR3 as determined by co-immunoprecipitation assays using lysates from transfected cells and by GST pull down assays using GST-Bcl-2-family fusion proteins. In contrast, Bcl-XL, Bcl-W, and Mcl-1 displayed little or no binding to TR3 (Nur77). Co-localization experiments using fluorescent protein tagging and confocal microscopy corroborated these findings, showing co-localization of a fragment of TR3 (Nur77) that accumulates in cytosol (rather than nucleus) [TR3 lacking the DNA-binding domain [DDBD]) with Bcl-2, Bfl-1, and Bcl-B on mitochondria and other intracellular organelles in intact cells, but not co-localization with Bcl-XL, Mcl-1 or Bcl-W. Co-expression of Bcl-2, Bfl-1, or Bcl-B with TR3DDBD by transfection resulted in robust apoptosis induction, while co-expression of Bcl-XL, Bcl-W, or Mcl-1 with TR3DDBD did not. In contrast to results obtained in TR3DDBD co-expression studies, expressing any of the anti-apoptotic Bcl-2-family proteins (Bcl-2, Bfl-1, Bcl-B, Bcl-XL, Mcl-1, Bcl-W) individually resulted in suppression of apoptosis induced by Staurosporine, illustrating the role of TR3 in converting the phenotypes of Bcl-2, Bfl-1 and Bcl-B from protector to killer. Because binding of TR3 (Nur77) to Bcl-B appeared to be strongest among the Bcl-2-family proteins, we focused on this Bcl-B for additional studies. (Ke, N. et al. J. Biol Chem276: 12481, 2001. Using monospecific antibodies, the endogenous Bcl-B protein was localized in human tissues, revealing predominant expression in plasma cells. Several myeloma cell lines also expressed Bcl-B protein, as determined by immunoblotting. Stimulating myeloma cell line RPMI8226 with ionomycin and phorbol ester TPA (agents that induce TR3 expression and accumulation in cytosol) resulted in association of endogenous TR3 with endogenous Bcl-B, as determined by co-immunoprecipitation experiments. Reducing endogenous Bcl-B levels using small interfering RNA (siRNA) reduced apoptosis induced by transfected TR3DDBD as well as apoptosis induced by a synthetic peptide that mimics TR3. We conclude that cytosolic TR3 (Nur77) interacts selectively with certain anti-apoptotic members of the Bcl-2-family (Bcl-2, Bfl-1, Bcl-B), converting them from protectors to killers. The ability of TR3 (Nur77) to convert Bcl-B suggests a possible novel strategy for triggering apoptosis of Bcl-B-expressing cells, which may be of utility for eradicating long-lived autoantibody-producing plasma cells or for killing malignant myeloma cells. (Supported by NIH GM60554 and SASS Foundation).

Description: An inverse relationship between BCL-2 expression and cell cycle transition has been suggested by recent studies in murine models. To investigate the clinical relevance of these laboratory studies, a group of 116 paraffin-embedded non-Hodgkin's lymphoma (NHL) biopsy specimens (Working Formulation Groups D-H, and J) from a cooperative group study of cellular DNA content were analyzed for the 14;18 translocation using polymerase chain reaction (PCR)-based methods and, if sufficient tissue remained, for BCL-2 and BAXexpression by immunohistochemistry. The results of these studies were then compared with the results of the previously performed flow cytometric analysis of ploidy and proliferative activity (S-phase-fraction). BCL-2 expression was inversely associated with proliferative activity (P = .001; n = 41), but there was no association between staining for Bax and %S-phase. Ploidy was not associated with either BCL-2 or BAX expression. The t(14;18) was detected in 21 of the 54 cases in which PCR-amplifiable DNA was recovered; 20 of these occurred at the major breakpoint region and 1 at the minor breakpoint region. High levels of BCL-2 orBAX expression occurred independently of t(14;18). There was no association between t(14;18) and either ploidy or proliferative activity. The inverse relationship between BCL-2 expression and proliferative activity in the intermediate- and high-grade NHLs is consistent with recent studies suggesting that Bcl-2 both retards entry into the cell cycle and inhibits apoptosis.

Description: B-cell chronic lymphocytic leukemia (B-CLL) represents a neoplastic disorder caused primarily by defective programmed cell death (PCD), as opposed to increased cell proliferation. Defects in the PCD pathway also contribute to chemoresistance. The expression of several apoptosis-regulating proteins, including the Bcl-2 family proteins Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, and BAD; the Bcl-2–binding protein BAG-1; and the cell death protease Caspase-3 (CPP32), was evaluated by immunoblotting using 58 peripheral blood B-CLL specimens from previously untreated patients. Expression of Bcl-2, Mcl-1, BAG-1, Bax, Bak, and Caspase-3 was commonly found in circulating B-CLL cells, whereas the Bcl-XL and BAD proteins were not present. Higher levels of the anti-apoptotic protein Mcl-1 were strongly correlated with failure to achieve complete remission (CR) after single-agent therapy (fludarabine or chlorambucil) (P = .001), but the presence of only seven CRs among the 42 patients for whom follow-up data were available necessitates cautious interpretation of these observations. Higher levels of the anti-apoptotic protein BAG-1 were also marginally associated with failure to achieve CR (P = .04). Apoptosis-regulating proteins were not associated with patient age, sex, Rai stage, platelet count, hemoglobin (Hb) concentration, or lymph node involvement, although higher levels of Bcl-2 and a high Bcl-2:Bax ratio were correlated with high numbers (〉105/μL) of white blood cells (WBC) (P = .01; .007) and higher levels of Bak were weakly associated with loss of allelic heterozygosity at 13q14 (P = .04). On the basis of measurements of apoptosis induction by fludarabine using cultured B-CLL specimens, in vitro chemosensitivity data failed to correlate with in vivo clinical response rates (n = 42) and expression of the various apoptosis-regulating proteins. Although larger prospective studies are required before firm conclusions can be reached, these studies show the expression in B-CLLs of multiple apoptosis-regulating proteins and suggest that the relative levels of some of these, such as Mcl-1, may provide information about in vivo responses to chemotherapy. In vitro chemosensitivity data, however, do not appear to be particularly useful in predicting responses in B-CLL.

Description: Defects in apoptosis mechanisms play important roles in malignancy and autoimmunity. Orphan nuclear receptor Nur77/TR3 has been demonstrated to bind antiapoptotic protein Bcl-2 and convert it from a cytoprotective to a cytodestructive protein, representing a phenotypic conversion mechanism. Of the 6 antiapoptotic human Bcl-2 family members, we found that Nur77/TR3 binds strongest to Bcl-B, showing selective reactivity with Bcl-B, Bcl-2, and Bfl-1 but not Bcl-XL, Mcl-1, or Bcl-W. Nur77 converts the phenotype of Bcl-B from antiapoptotic to proapoptotic. Bcl-B is prominently expressed in plasma cells and multiple myeloma. Endogenous Bcl-B associates with endogenous Nur77 in RPMI 8226 myeloma cells, where RNA interference experiments demonstrated dependence on Bcl-B for Nur77-induced apoptosis. Furthermore, a Nur77-mimicking peptide killed RPMI 8226 myeloma cells through a Bcl-B–dependent mechanism. Because Bcl-B is abundantly expressed in plasma cells and some myelomas, these findings raise the possibility of exploiting the Nur77/Bcl-B mechanism for apoptosis for eradication of autoimmune plasma cells or myeloma.

Description: p53, a key regulator of apoptosis, functions primarily upstream in the apoptotic cascade by directly and indirectly modulating Bcl-2 family of proteins. XIAP, a potent antiapoptotic protein, functions primarily downstream by suppressing caspases. Activation of p53 by MDM2 antagonist nutlin3a or inhibition of XIAP by small molecule inhibitors such as phenylurea 1396-11 was found to induce apoptosis in AML cells. Since the functions of XIAP and p53 are mediated and their activities controlled by a network of numerous components, some of which cross-regulate each other, we hypothesized that simultaneous activation of p53 and inhibition of XIAP would be a more effective at activating apoptotic signaling in AML cells. To test this idea, we treated AML cells with nutlin3a and 1396-11 and found that the combination synergistically induced cell death at 24 hours in OCIAML3 cells (combination index CI=0.200±0.047) and Molm13 cells (CI=0.565±0.082), two cell lines harboring wild type p53. Knocking down p53 expression by shRNA blunted the synergistic effect and downregulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin3a-induced apoptosis in OCI-AML3 cells, suggesting that the synergy was mediated by both p53 activation and XIAP inhibition. The specificity was further supported by data showing that inhibition of MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Although nutlin3a alone induced apoptosis in OCI-AML3 cells, the cell death was not robust and caspase-3 activation was minimal by itself even at 48 hours with 10 μM of nutlin3a. Immunoblot analysis showed increased expression of p53 and its downstream target p21. Of note, because p21 not only induces G1 cell cycle block, it additionally exhibits antiapoptotic activity that diminishes the effects of p53 activation, we also studied effects of these agents on p21 levels. When nutlin3a and 1396–11 were combined, caspase-3 activation was greatly increased and nutlin3a-induced p21 expression was significantly diminished. Moreover, in these experiments, caspase inhibition restored p21 levels and diminished apoptosis enhanced by 1396-11, suggesting that XIAP inhibition-mediated caspase activation eliminates p21, enhancing nutlin3a-induced apoptosis. Furthermore, activation of p53 by nutlin3a increased caspase-6 protein levels and induced mitochondrial release of SMAC, an antagonist of XIAP, suggesting that p53 activation shifts the balance toward apoptosis, promoting the effect of XIAP inhibition. Most importantly, p53 activation and XIAP inhibition greatly enhanced apoptosis in primary blasts from AML patients. Five out of six samples treated showed synergistic killing at 24 hours (CI=0.73±0.13), even when the cells were protected from drug-induced and spontaneous apoptosis by MS-5 stroma cells (CI=0.45±0.06). In conclusion, results demonstrate that simultaneous activation of p53 by antagonizing MDM2 and inhibition of XIAP synergistically activate apoptotic signaling pathways and promote death of AML cells, in part by modulating p21, caspases, and cytosolic SMAC levels. Since both, XIAP and p53, are presently being targeted by ongoing clinical trials in leukemia patients, the combination strategy holds promise for expedited translation into the clinic.

Description: We tested the effects of small-molecule XIAP antagonists based on a polyphenylurea pharmacophore on cultured acute myelogenous leukemia (AML) cell lines and primary patient samples. X-linked inhibitor of apoptosis protein (XIAP) antagonist N-[(5R)-6-[(anilinocarbonyl)amino]-5-((anilinocarbonyl){[(2R)-1-(4-cyclohexylbutyl)pyrrolidin-2-yl]methyl}amino)hexyl]-N-methyl-N′-phenylurea (1396-12), but not a structurally related control compound, induced apoptosis of primary leukemia samples with a lethal dose (LD50) of less than 10 μM in 16 of 27 (60%) samples. In contrast, XIAP antagonist 1396-12 was not lethal to the normal hematopoietic cells in short-term cytotoxicity assays. Response of primary AML specimens to XIAP inhibitor correlated with XIAP protein levels, with higher levels of XIAP associated with sensitivity. The XIAP antagonist 1396-12 induced activation of downstream caspases 3 and 7 prior to the activation of upstream caspase 8 and caspase 9. Apoptosis induction was also independent of B-cell lymphoma protein-2 (Bcl-2) or caspase 8, indicative of a downstream effect on apoptotic pathways. Thus, polyphenylurea-based XIAP antagonsists directly induce apoptosis of leukemia cells and AML patient samples at low micromolar concentrations through a mechanism of action distinct from conventional chemotherapeutic agents.

Description: We tested the effects of Rituximab (anti-CD20) and IDEC-152 (anti-CD23) on apoptosis of B-cell malignancies, using established non-Hodgkin’s B-Cell lymphoma cell lines and freshly isolated Chronic Lymphocytic Leukemia (CLL) B-cells. We used monolayers of stably transfected CHO-cells expressing FcRγIII-A to present antibody to B-cells and promote crosslinking. Established B-cell lymphomas (n = 3) were cultured in the presence of FcRγIIIA-expressing CHO monolayer with or without MAbs and apoptosis was measured by annexin V/propidium iodide staining at various times thereafter. Both antibodies induced time-dependent apoptosis of B-cell lymphoma cell lines. After 48 hrs of treatment with either Rituximab or IDEC-152, the majority of the malignant B-cells were apoptotic (remaining viable cells = 28.7% ± 0.2137% for Rituximab and 30.87% ± 0.7332% for IDEC-152). Rituximab and IDEC-152 also induced marked increases in caspase activity in B-cell lymphoma cell lines, with fold-increases above baseline control cells of 25 ± 0.9031 and 24 ± 0.3839, respectively. In contrast, neither Rituximab nor IDEC-152 induced striking effects on primary CLL B-cells (n = 6). We therefore tested the combination of Rituximab or IDEC-152 with other agents that target anti-apoptotic proteins, exploring whether more efficient induction of apoptosis can be achieved. We cultured lymphoma cell lines and primary CLL specimens with chemical antagonists of XIAP (Schimmer, et al. Cancer Cell5: 25, 2004), an anti-apoptotic protein that inhibits effector caspases. When used at concentrations where XIAP antagonists alone were non-apoptotic (approximately 2.5 μM), a significant increase in apoptosis was achieved in cultures of lymphoma and CLL cells treated with either Rituximab or IDEC-152. These findings suggest that Rituximab or IDEC-152 may more efficiently induce apoptosis of malignant B-cells when combined with an apoptosis-sensitizing agent. (Supported by CA-81534; CA-78040; and an unrestricted grant from Genentech, Inc.).

Description: Chronic lymphocytic leukemia (CLL) cells develop chemo-resistance over time. Most anticancer agents function through induction of apoptosis, and therefore resistance against these agents is likely to be caused by selection for CLL cells with defects in the particular apoptosis pathway that is triggered by these drugs. Anticancer agents that function through alternative apoptotic pathways might therefore be useful in treating chemo-resistant CLL. Triterpenoids represent a class of naturally occurring and synthetic compounds with demonstrated antitumor activity. We examined the effects of CDDO (triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid) on CLL B cells in vitro. CDDO induced apoptosis in a dose-dependent manner in all (n = 30) CLL samples tested, including previously untreated and chemo-resistant CLL specimens. CDDO induced rapid proteolytic processing of caspase-8, but not caspase-9, in CLL B cells, suggesting activation of a mitochondria-independent pathway. CDDO-induced apoptosis of CLL B cells was blocked by cytokine response modifier A (CrmA), a suppressor of caspase-8, but not by X-linked inhibitor of apoptosis protein–baculovirus IAP repeat–3 (XIAP-BIR3), a fragment of XIAP, which selectively inhibits caspase-9. Examination of CDDO effects on expression of several apoptosis-relevant genes demonstrated significant reductions in the levels of caspase-8 homolog Fas-ligand interleukin-1–converting enzyme (FLICE)–inhibitory protein (c-FLIP), an endogenous antagonist of caspase-8. However, reductions of FLIP achieved by FLIP antisense oligonucleotides were insufficient for triggering apoptosis, indicating that CDDO has other targets in CLL B cells besides FLIP. These data suggest that the synthetic triterpenoid CDDO should be further explored as a possible therapeutic agent for treatment of chemo-resistant CLL.

Description: Abstract 2421 Members of the nuclear factor-kB (NF-κB) family of transcription factors play important roles in cell-signaling involved in host-defense, immune responses, inflammation, and cancer. Multiple stimuli operating through different receptors are capable of activating NF-κB, including the Tumor Necrosis Factor (TNF) family cytokines, Toll-like receptors (TLRs), Nucleotide-binding Leucine Rich Repeat Proteins (NLRs), DNA-damaging agents, and others. Drugs that selectively block activation of NF-κB through one or more of these receptors could have activity against various types of cancer in which activation of NF-κB plays a role. We used a chemical biology strategy to identify novel chemical inhibitors of cell pathways responsible for NF-κB activation. Screening of a large chemical library (〉330K compounds from NIH) was conducted using human 697 pre-B-cell leukemia cells with a stably integrated NF-κB-luciferase reporter gene and stimulating the Carma/Bcl-10/MALT pathway using protein kinase C (PKC) activators (PMA/Ionomycin). Confirmed hits were counter-screened against HEK293T-NF-κB-luciferase cells treated with TNF-a, with the goal to eliminate non-pathway specific inhibitors of NF-κB. Among the hit compounds, we identified oxadiazole- and oxazole-based chemical probes as potential cell lineage- and cell differentiation-specific inhibitors of NF-κB (“NLDSi” = NF-κB lineage, differentiation-specific inhibitors). Using a panel of cell lines with stably integrated NF-κB-driven luciferase reporter genes, NLDSi compounds suppressed PMA/Ionomycin-induced NF-κB activity in a concentration-dependent manner (IC50=0.1–1.0 μM) in the pre-B-cell leukemia cell-line 697, but not in the T-cell leukemia line JURKAT, the myeloma cell-line RPMI8226, or HEK293 epithelial cells. These compounds also suppressed NF-κB activity (measured by reporter gene) in 697 cells stimulated with BAFF or treated with doxorubicin. Examining NF-κB target gene expression by q-RT-PCR (mRNA level) and immunoblotting (protein level) showed that NLDSi compounds reduced PMA/Ionomycin-induced TRAF1 and A20 expression in 697 cells and in the pre-B-cell leukemia cell line REH, but not in several mature B-cell lines, including BJAB, Daudi, OCILy3, and OCILy10. NLDSi compounds also inhibited NF-κB target gene expression in pre-B-cell lines stimulated with BAFF or treated with lipopolysaccharide (LPS) or doxorubicin (Dox). Nevertheless we found these compounds could suppress of PMA/Ionomycin-induced expression of TRAF1 and A20 gene in the primary leukemia cells of patients with chronic lymphocytic leukemia (CLL). Taken together, the compounds described here appear to selectively suppress NF-κB activation in the B-cell lineage at specific stages of differentiation. As such, these small molecules could serve as chemical probes for uncovering novel targets that regulate NF-κB activity and potentially lead to development of therapies that selectively block certain types of NF-κB activation in pre-B cell ALL, CLL, or other hematologic malignancies. (Supported by NIH grants X01 MH077633-1, U54–005033, and P01-CA-81534). Disclosures: No relevant conflicts of interest to declare.