nitrophenols and Lymphoma--Large-B-Cell--Diffuse

GNA13, encoding one of the G protein alpha subunits of heterotrimeric G proteins that transduce signals of G protein-coupled receptors (GPCR), is frequently mutated in germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) with poor prognostic outcomes. Due to the "undruggable" nature of GNA13, targeted therapy for these patients is not available. In this study, we found that palmitoylation of GNA13 not only regulates its plasma membrane localization, but also regulates GNA13's stability. It is essential for the tumor suppressor function of GNA13 in GCB-DLBCL cells. Interestingly, GNA13 negatively regulates BCL2 expression in GCB-DLBCL cells in a palmitoylation-dependent manner. Consistently, BCL2 inhibitors were found to be effective in killing GNA13-deficient GCB-DLBCL cells in a cell-based chemical screen. Furthermore, we demonstrate that inactivating GNA13 by targeting its palmitoylation enhanced the sensitivity of GCB-DLBCL to the BCL2 inhibitor. These studies indicate that the loss-of-function mutation of GNA13 is a biomarker for BCL2 inhibitor therapy of GCB-DLBCL and that GNA13 palmitoylation is a potential target for combination therapy with BCL2 inhibitors to treat GCB-DLBCL with wild-type GNA13.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Female; GTP-Binding Protein alpha Subunits, G12-G13; HeLa Cells; Humans; Lipoylation; Lymphoma, Large B-Cell, Diffuse; Male; Mice; Mice, Inbred NOD; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

To investigate the roles of BCL2, MCL1, and BCL-XL in the survival of diffuse large B-cell lymphoma (DLBCL).. Immunohistochemical analysis of 105 primary DLBCL samples, and Western blot analysis of 18 DLBCL cell lines for the expression of BCL2, MCL1, and BCL-XL. Pharmacologic targeting of BCL2, MCL1, and BCL-XL with ABT-199, homoharringtonine (HHT), and ABT-737. Analysis of DLBCL clones with manipulated expressions of BCL2, MCL1, and BCL-XL. Immunoprecipitation of MCL1 complexes in selected DLBCL cell lines. Experimental therapy aimed at inhibition of BCL2 and MCL1 using ABT-199 and HHT, single agent, or in combination, in vitro and in vivo on primary cell-based murine xenograft models of DLBCL.. By the pharmacologic targeting of BCL2, MCL1, and BCL-XL, we demonstrated that DLBCL can be divided into BCL2-dependent and MCL1-dependent subgroups with a less pronounced role left for BCL-XL. Derived DLBCL clones with manipulated expressions of BCL2, MCL1, and BCL-XL, as well as the immunoprecipitation experiments, which analyzed MCL1 protein complexes, confirmed these findings at the molecular level. We demonstrated that concurrent inhibition of BCL2 and MCL1 with ABT-199 and HHT induced significant synthetic lethality in most BCL2-expressing DLBCL cell lines. The marked cytotoxic synergy between ABT-199 and HHT was also confirmed in vivo using primary cell-based murine xenograft models of DLBCL.. As homoharringtonine is a clinically approved antileukemia drug, and ABT-199 is in advanced phases of diverse clinical trials, our data might have direct implications for novel concepts of early clinical trials in patients with aggressive DLBCL.

Topics: Animals; Apoptosis; bcl-X Protein; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Harringtonines; Homoharringtonine; Humans; Lymphoma, Large B-Cell, Diffuse; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Xenograft Model Antitumor Assays

The BCL6 oncogene plays a crucial role in sustaining diffuse large B-cell lymphomas (DLBCL) through transcriptional repression of key checkpoint genes. BCL6-targeted therapy kills lymphoma cells by releasing these checkpoints. However BCL6 also directly represses several DLBCL oncogenes such as BCL2 and BCL-XL that promote lymphoma survival. Herein we show that DLBCL cells that survive BCL6-targeted therapy induce a phenomenon of "oncogene-addiction switching" by reactivating BCL2-family dependent anti-apoptotic pathways. Thus, most DLBCL cells require concomitant inhibition of BCL6 and BCL2-family members for effective lymphoma killing. Moreover, in DLBCL cells initially resistant to BH3 mimetic drugs, BCL6 inhibition induces a newly developed reliance on anti-apoptotic BCL2-family members for survival that translates in acquired susceptibility to BH3 mimetic drugs ABT-737 and obatoclax. In germinal center B cell-like (GCB)-DLBCL cells, the proteasome inhibitor bortezomib and the NEDD inhibitor MLN4924 post-transcriptionally activated the BH3-only sensitizer NOXA thus counteracting the oncogenic switch to BCL2 induced by BCL6-targeting. Hence our study indicates that BCL6 inhibition induces an on-target feedback mechanism based on the activation of anti-apoptotic BH3 members. This oncogene-addition switching mechanism was harnessed to develop rational combinatorial therapies for GCB-DLBCL.

Topics: Animals; Biphenyl Compounds; Blotting, Western; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Indoles; Lymphoma, Large B-Cell, Diffuse; Male; Mice; Mice, Nude; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-bcl-6; Pyrroles; Rats; Rats, Sprague-Dawley; RNA, Small Interfering; Sulfonamides; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Previously, we have demonstrated that inhibition of Hedgehog pathway induces predominantly apoptosis in diffuse large B-cell lymphoma (DLBCL) cell lines of activated B-cell (ABC) type but predominantly cell cycle arrest in those of germinal center (GC). Here, we explored the possibility of overcoming the resistance to apoptosis to SMO inhibitors in five DLBCL cells of GC type using the combination of the SMO inhibitor HhAntag (Genentech Inc) with the BH3 mimetic ABT-737 (Abbott Laboratories). As controls we have used two DLBCL of ABC type (OCI-LY10 and OCI-LY3). Combinatorial treatments were performed with increasing concentrations of the HhAntag with low doses (equal or less than the IC20) of ABT-737. MTS assays were used to detect changes in cell viability and Annexin-V and PARP1 cleavage assays were used to detect apoptosis. Combining low doses of ABT-737 with increasing concentrations of HhAntag in GC DLBCL cell lines resulted in significantly increase of apoptosis in comparison to treatments with the SMO inhibitor alone. We concluded that in GC DLBCL cell lines, in contrast to those of ABC type, functional inhibition of BCL2 family members is usually needed to overcome the resistance to apoptosis to SMO inhibitors. These findings provide a rationale to explore the use of SMO and BCL2 inhibitors as adjuvant therapy for treatment of DLBCL of GC type.

Topics: Anilides; Apoptosis; Apoptosis Regulatory Proteins; Biphenyl Compounds; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Germinal Center; Humans; Inhibitory Concentration 50; Lymphoma, Large B-Cell, Diffuse; Neoplasm Proteins; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Sulfonamides

BCL2 suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating outer mitochondrial membrane permeabilization. Many tumor types, including B-cell lymphomas and chronic lymphocytic leukemia, are dependent on BCL2 for survival but become resistant to apoptosis after treatment. Here, we identified a direct interaction between the antiapoptotic protein BCL2 and the enzyme PARP1, which suppresses PARP1 enzymatic activity and inhibits PARP1-dependent DNA repair in diffuse large B-cell lymphoma cells. The BH3 mimetic ABT-737 displaced PARP1 from BCL2 in a dose-dependent manner, reestablishing PARP1 activity and DNA repair and promoting nonapoptotic cell death. This form of cell death was unaffected by resistance to single-agent ABT-737 that results from upregulation of antiapoptotic BCL2 family members. On the basis of the ability of BCL2 to suppress PARP1 function, we hypothesized that ectopic BCL2 expression would kill PARP inhibitor-sensitive cells. Strikingly, BCL2 expression reduced the survival of PARP inhibitor-sensitive breast cancer and lung cancer cells by 90% to 100%, and these effects were reversed by ABT-737. Taken together, our findings show that a novel interaction between BCL2 and PARP1 blocks PARP1 enzymatic activity and suppresses PARP1-dependent repair. Targeted disruption of the BCL2-PARP1 interaction therefore may represent a potential therapeutic approach for BCL2-expressing tumors resistant to apoptosis.

Topics: Animals; Biphenyl Compounds; Cell Death; Cell Line, Tumor; Cell Nucleus; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Large B-Cell, Diffuse; Methylnitronitrosoguanidine; Mice; Nitrophenols; Piperazines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Most cancer cells utilize aerobic glycolysis, and activation of the phosphoinositide 3-kinase/Akt/mTOR pathway can promote this metabolic program to render cells glucose dependent. Although manipulation of glucose metabolism may provide a means to specifically eliminate cancer cells, mechanistic links between cell metabolism and apoptosis remain poorly understood. Here, we examined the role and metabolic regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in cell death upon inhibition of Akt-induced aerobic glycolysis. In the presence of adequate glucose, activated Akt prevented the loss of Mcl-1 expression and protected cells from growth factor deprivation-induced apoptosis. Mcl-1 associated with and inhibited the proapoptotic Bcl-2 family protein Bim, contributing to cell survival. However, suppression of glucose metabolism led to induction of Bim, decreased expression of Mcl-1, and apoptosis. The proapoptotic Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, shows clinical promise, but Mcl-1 upregulation can promote resistance. Importantly, inhibition of glucose metabolism or mTORC1 overcame Mcl-1-mediated resistance in diffuse large B cell leukemic cells. Together these data show that Mcl-1 protein synthesis is tightly controlled by metabolism and that manipulation of glucose metabolism may provide a mechanism to suppress Mcl-1 expression and sensitize cancer cells to apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Adenosine Triphosphate; Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Biphenyl Compounds; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Neoplastic; Glucose; Glycolysis; Humans; Jurkat Cells; Lymphoma, Large B-Cell, Diffuse; Mechanistic Target of Rapamycin Complex 1; Membrane Proteins; Mice; Multiprotein Complexes; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitrophenols; Phosphoproteins; Piperazines; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Ribosomal Protein S6 Kinases; Sulfonamides; T-Lymphocytes; TOR Serine-Threonine Kinases

ABT-737 is a small-molecule antagonist of BCL-2 currently under evaluation in clinical trials in the oral form of ABT-263. We anticipate that acquired resistance to this promising drug will inevitably arise. To study potential mechanisms of resistance to ABT-737, we derived resistant lines from initially sensitive OCI-Ly1 and SU-DHL-4 lymphoma cell lines via long-term exposure. Resistance was based in the mitochondria and not due to an inability of the drug to bind BCL-2. Resistant cells had increased levels of BFL-1 and/or MCL-1 proteins, which are not targeted by ABT-737. Proapoptotic BIM was displaced from BCL-2 by ABT-737 in both parental and resistant cells, but in resistant cells, BIM was sequestered by the additional BFL-1 and/or MCL-1. Decreasing MCL-1 levels with flavopiridol, PHA 767491, or shRNA restored sensitivity to ABT-737 resistant cells. MCL-1 was up-regulated not by protein stabilization but rather by increased transcript levels. Surprisingly, in addition to stable increases in MCL-1 transcript and protein in resistant cells, there was a dynamic increase within hours after ABT-737 treatment. BFL-1 protein and transcript levels in resistant cells were similarly dynamically up-regulated. This dynamic increase suggests a novel mechanism whereby modulation of antiapoptotic protein function communicates with nuclear transcriptional machinery.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; BH3 Interacting Domain Death Agonist Protein; Biphenyl Compounds; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression; Gene Expression Profiling; Humans; Immunoblotting; Immunoprecipitation; Lymphoma, Large B-Cell, Diffuse; Membrane Proteins; Minor Histocompatibility Antigens; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides; Up-Regulation

Overexpression of antiapoptotic members of the Bcl-2 family is observed in approximately 80% of B-cell lymphomas, contributing to intrinsic and acquired drug resistance. Nullifying the antiapoptotic influence of these proteins can potentially overcome this resistance, and may complement conventional chemotherapy. ABT-737 is a BH3-only mimetic and potent inhibitor of the antiapoptotic Bcl-2 family members Bcl-2, Bcl-X(L), and Bcl-w. In vitro, ABT-737 exhibited concentration-dependent cytotoxicity against a broad panel of lymphoma cell lines including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). ABT-737 showed synergism when combined with the proteasome inhibitors bortezomib or carfilzomib in select lymphoma cell lines and induced potent mitochondrial membrane depolarization and apoptosis when combined with either. ABT-737 plus bortezomib also induced significant apoptosis in primary samples of MCL, DLBCL, and chronic lymphocytic leukemia (CLL) but no significant cytotoxic effect was observed in peripheral blood mononuclear cells from healthy donors. In severe combined immunodeficient beige mouse models of MCL, the addition of ABT-737 to bortezomib enhanced efficacy compared with either drug alone and with the control. Collectively, these data suggest that ABT-737 alone or in combination with a proteasome inhibitor represents a novel and potentially important platform for the treatment of B-cell malignancies.

Topics: Animals; Antineoplastic Agents; Biphenyl Compounds; Boronic Acids; Bortezomib; Cell Death; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; Health; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukocytes, Mononuclear; Lymphoma; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Mantle-Cell; Membrane Potential, Mitochondrial; Mice; Microscopy, Confocal; Molecular Mimicry; Nitrophenols; Piperazines; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Sulfonamides; Tissue Donors; Xenograft Model Antitumor Assays

Cancer cells exhibit many abnormal phenotypes that induce apoptotic signaling via the intrinsic, or mitochondrial, pathway. That cancer cells nonetheless survive implies that they select for blocks in apoptosis. Identifying cancer-specific apoptotic blocks is necessary to rationally target them. Using a panel of 18 lymphoma cell lines, we show that a strategy we have developed, BH3 profiling, can identify apoptotic defects in cancer cells and separate them into three main classes based on position in the apoptotic pathway. BH3 profiling identifies cells that require BCL-2 for survival and predicts sensitivity to the BCL-2 antagonist ABT-737. BCL-2 dependence correlates with high levels of proapoptotic BIM sequestered by BCL-2. Strikingly, BH3 profiling can also predict sensitivity to conventional chemotherapeutic agents like etoposide, vincristine, and adriamycin.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; BH3 Interacting Domain Death Agonist Protein; Biphenyl Compounds; Blotting, Western; Doxorubicin; Etoposide; Humans; Immunoblotting; Immunoprecipitation; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Membrane Proteins; Nitrophenols; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Tumor Cells, Cultured; Vincristine