gx-15-070 and Lymphoma--Large-B-Cell--Diffuse

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

Interactions between the irreversible proteasome inhibitor carfilzomib and the pan-BH3 mimetic obatoclax were examined in germinal center (GC)- and activated B-cell-diffuse large B-cell lymphoma (ABC-DLBCL) cells. Cotreatment with minimally toxic concentrations of carfilzomib (i.e., 2-6 nmol/L) and subtoxic concentrations of obatoclax (0.05-2.0 μmol/L) synergistically increased apoptosis in multiple DLBCL cell lines and increased lethality toward primary human DLBCL but not normal CD34(+) cells. Synergistic interactions were associated with sharp increases in caspase-3 activation, PARP cleavage, p-JNK induction, upregulation of Noxa, and AKT dephosphorylation. Combined treatment also diminished carfilzomib-mediated Mcl-1 upregulation whereas immunoprecipitation analysis revealed reduced associations between Bak and Mcl-1/Bcl-xL and Bim and Mcl-1. The carfilzomib/obatoclax regimen triggered translocation, conformational change, and dimerization of Bax and activation of Bak. Genetic interruption of c-jun-NH(2)-kinase (JNK) and Noxa by short hairpin RNA knockdown, ectopic Mcl-1 expression, or enforced activation of AKT significantly attenuated carfilzomib/obatoclax-mediated apoptosis. Notably, coadministration of carfilzomib/obatoclax sharply increased apoptosis in multiple bortezomib-resistant DLBCL models. Finally, in vivo administration of carfilzomib and obatoclax to mice inoculated with SUDHL4 cells substantially suppressed tumor growth, activated JNK, inactivated AKT, and increased survival compared with the effects of single-agent treatment. Together, these findings argue that a strategy combining carfilzomib and obatoclax warrants attention in DLBCL.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Cell Line, Tumor; Drug Synergism; Germinal Center; Histones; Humans; Indoles; Lymphoma, Large B-Cell, Diffuse; MAP Kinase Kinase 4; Membrane Proteins; Mice; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Xenograft Model Antitumor Assays

Bcl-2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3-mimetic, has been designed to specifically target and counteract anti-apoptotic Bcl-2 proteins. We evaluated the biological effects of obatoclax on the anti-tumour activity of rituximab and chemotherapy agents. Obatoclax induced cell death of rituximab/chemotherapy-sensitive (RSCL), -resistant cell lines (RRCL) and primary tumour-cells derived from patients with B-cell lymphomas (N=39). Obatoclax also enhanced the activity of rituximab and had synergistic activity when combined with chemotherapy agents. The ability of Obatoclax to induce PARP cleavage varied between patient samples and was not observed in some RRCL. Inhibition of caspase activity did not affect obatoclax activity, suggesting the existence of caspase-independent death pathways. Autophagy was detected by LC3 conversion and/or electron microscopy in RRCL and in patient-derived tumour cells. Moreover, obatoclax activity was inhibited by Beclin-1 knockdown. In summary, obatoclax is an active Bcl-2 inhibitor that potentiates the activity of chemotherapy agents and, to a lesser degree, rituximab. Defining the molecular events triggered by obatoclax is necessary to further its clinical development and identify potential biomarkers that are predictive of response.

Topics: Antibodies, Monoclonal, Murine-Derived; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Combined Chemotherapy Protocols; Apoptosis Regulatory Proteins; Autophagy; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Caspases; Cell Death; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Drug Synergism; Humans; Indoles; Lymphoma, B-Cell; Lymphoma, Follicular; Lymphoma, Large B-Cell, Diffuse; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Rituximab; Tumor Cells, Cultured; Up-Regulation