Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse and human c-MYC-transformed lymphomas reveals IKK2/NF-kB-pathway suppresses MYC-induced lymphomagenesis

ABSTRACT: Abstract. Deregulated c-MYC is found in a variety of cancers where it promotes proliferation as well as apoptosis. In many hematological malignancies enhanced NF-kB exerts prosurvival functions. Here we investigated the role of NF-kB in mouse and human c-MYC-transformed lymphomas. The NF-kB-pathway is extinguished in murine lymphoma cells and extrinsic stimuli typically inducing NF-kB activity fail to activate this pathway. Genetic activation of the NF-kB pathway induces apoptosis in these cells, while inhibition of NF-kB by an IkBa superrepressor provides a selective advantage in vivo. Furthermore, in human BurkittÂ´s lymphoma cells we find that NF-kB activation induces apoptosis. NF-kB upregulates Fas and predisposes to Fas-induced cell death, which is caspase 8 mediated and can be prevented by CFLAR overexpression. We conclude that c-MYC overexpression sensitizes cells to NF-kB-induced apoptosis and persistent inacvtivity of NF-kB signaling is a prerequisite for MYC-mediated tumorigenesis. We could also show that low immunogenicity and Fas insensitivity of MYC driven lymphoma cells is reversed by activation of NF-kB. Our observations provide a molecular explanation for the described absence of the NF-kB signaling in BurkittÂ´s lymphoma and question the applicability of NF-kB inhibitors as candidates for treatment of this cancer. Experiment Overall Design: Ramos cells were transfected in triplicates with pRTS-GFP (A+,C+,D+) or pRTS-CA-IKK2 (E+,F+,H+), selected with hygromycin and transgene expression was induced with doxycycline (0,5mg/ml) for 48hrs. RNA was isolated with RNeasy mini kit (Qiagen, Venlo, Netherlands) and Gene expression profiling (GEP) was performed using Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA). 2 Âµg of total RNA were labeled using the GeneChipÂ® One-Cycle Target Labeling assay kit (Affymetrix). After hybridization arrays were stained and washed in a FS 450 Fluidics station (Affymetrix) before imaging on an Affymetrix GeneChip (3000) scanner. Raw data were generated using the GCOS 1.4 software (Affymetrix). Probe level data were obtained using the Robust Multichip Average (RMA) normalization algorithm and CEL files were loaded into Genesifter (GeneSifter.Net, VizX Laboratories, Seattle, WA, USA). Genes were identified as differentially expressed among the two classes if a two-sample T-test revealed a nominal significance level of 0.05 and the ratio between the two classes was at least 2 fold. Calculation of false discovery rate was done according to the method of Benjamini and Hochberg. Biological significance was determined using Gene Ontolgy reports.

ORGANISM(S): Homo sapiens

SUBMITTER: Kay Klapproth

PROVIDER: E-GEOD-17129 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Aberrant B-cell receptor (BCR)/NF-kB signaling activity is a prominent feature of diffuse large B-cell lymphoma (DLBCL), particularly of, but not restricted to the activated B-cell (ABC) subtype. Recurrent mutations in this cascade, e.g. in CD79B, CARD11, A20/TNFAIP3 or NFKBIZ, but also in the Toll-like receptor (TLR) pathway transducer MyD88, all converge at NF-kB deregulation, but their differential impact on lymphoma development and biology remains to be dissected. Recapitulating oncogenic myc rearrangements as another common feature of DLBCL, we functionally investigate here primary mouse lymphomas that formed after propagation of Eµ-myc transgenic hematopoietic stem cells (HSC), stably transduced with naturally occurring DLBCL-derived NF-kB mutants, in recipient mice. While most mutants tested supported Myc-driven lymphoma formation through repressed apoptosis, selectively deregulated CARD11- or MyD88-mutant lymphoma cells presented with a macrophage-activating secretion profile, which, in turn, enforced TGF-b-mediated feedback senescence in the lymphoma cell compartment. Moreover, MyD88- or CARD11-mutant Eµ-myc lymphomas – mirrored by matching signatures in their mutant DLBCL counterparts – exhibited high-level expression of the immune checkpoint mediator PD-L1, thus preventing their efficient clearance by adaptive host cell immunity. Conversely, these mutant-specific dependencies were therapeutically exploitable by anti-PD1 immune checkpoint blockade, leading to the direct T-cell-mediated lysis of predominantly but not exclusively senescent lymphoma cells. Our functional, cross-species interrogation of a syngeneic and fully immune-competent, BCR/NF-kB-deregulated and DLBCL-reminiscent in vivo-model platform unveils common principles and therapeutic vulnerabilities related to human DLBCL subsets that will inform future personalized treatment strategies. Specifically, for the shown FLC transplantation experiments, 8-12 week-old female C57BL/6N recipient mice (Charles River Laboratories) received a single, myelo-ablative dose of 9 Gy total-body gamma-irradiation. After in vitro-transduction of FLC, 10% GFP-positive Sca1+; c-Kit+; lin- cells with the indictaed transgenic and/or knock out genotypes were transplanted via tail vein injection into irradiated mice, which were subsequently monitored until a well-palpable lymphadenopathy had formed. Retroviral vectors used in this experiment were the MSCV 2.2 derived constructs

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Transcription profiling of mouse and human c-MYC-transformed lymphomas reveals IKK2/NF-kB-pathway suppresses MYC-induced lymphomagenesis

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