Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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IkB-like protein NFKBIZ regulates NF-kB signaling and is critical for survival of ABC DLBCL (NFKBIZ inhibition)

ABSTRACT: Constitutive activation of the nuclear factor-kappa B (NF-kB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-kB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the IkB-like protein NFKBIZ that binds NF-kB subunits and enhances transactivation of some NF-kB target genes while repressing others, to be upregulated in ACB compared to GCB DLBCL primary patient samples (p=5.1 x 10^-37). Knockdown of NFKBIZ by RNA interference was toxic to ABC but not GCB DLBCL cell lines. Gene expression profiling following NFKBIZ knockdown significantly downregulated a large number of NF-kB target genes, suggesting a central role in regulating NF-kB signaling. To further investigate the molecular mechanisms of how NFKBIZ mediates NF-kB signaling in ABC DLBCL, we performed immunoprecipitations and detected an interaction of NFKBIZ with both p50 and p52 NF-kB subunits, indicating that both the canonical and non-canonical NF-kB pathways are regulated by NFKBIZ. Collectively, our data imply that NFKBIZ is required for NF-kB signaling in ABC DLBCL and thus might represent a promising molecular target for future therapies. The complete dataset is comprised of three experiments with the male HBL-1 ABC DLBCL cell line: a) 8 paired GEP measurements after NFKBIZ inhibition by shRNA, b) 6 paired GEP measurements after applying the MLN inhibitor and c) 4 two-color measurements after applying a MALT inhibitor. This dataset includes 8 paired GEP measurements after NFKBIZ inhibition by shRNA.

ORGANISM(S): Homo sapiens

SUBMITTER: Michael Grau

PROVIDER: E-GEOD-46971 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The ABC subtype of diffuse large B cell lymphoma (DLBCL) remains the least curable form of this lymphoma despite recent advances in therapy. We have combined structural and functional genomics to triangulate on new oncogenic mechanisms and devise new therapeutic strategies. RNA interference screen revealed a dependence of ABC DLBCL cell lines on MYD88 and IRAK1. High throughput resequencing of RNA (RNA-Seq) revealed frequent somatic mutations in MYD88 that preferentially occurred in the ABC DLBCL subtype. Remarkably, one third of ABC DLBCL tumor samples harbored the same amino acid substitution, L265P, in the MYD88 TIR domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in two other DLBCL subtypes, but was observed in 9% of MALT lymphomas. At a lower frequency, multiple other mutations were observed in the MYD88 TIR domain, occurring in both the ABC and GCB subtypes of DLBCL. Survival of ABC DLBCL lines bearing the L265P mutation was sustained by the mutant but not wild type MYD88 isoform, demonstrating that this MYD88 mutant is oncogenic and gain-of-function. The MYD88 L265P mutant assembled a protein complex that spontaneous triggers the phosphorylation of IRAK1, leading to NF-kB signaling, secretion of the cytokines IL-6, IL-10 and interferon-b, and JAK kinase signaling. These findings demonstrate that the MYD88 signaling pathway is integral to the pathogenesis of ABC DLBCL, providing a genetic rationale for therapeutic targeting of the MYD88 signaling pathway in this lymphoma subtype. To generate a gene expression signature of MYD88 signaling in ABC DLBCL, the HBL-1 cell line was transduced with retroviral vectors expressing either shMYD88-4 or shMYD88-7. Following puromycin selection, shRNA expression was induced for 24 or 48 hours and gene expression was measured, comparing uninduced (Cy3) to induced (Cy5) cells, using genome-wide Agilent 4x44K oligonucleotide microarrays. A signature of NF-kB signaling in ABC DLBCL was generated by treating HBL-1 cells with the IkB kinase beta inhibitor MLN120B for 2h, 3h, 4h, 6h, 8h, 12h, 16h, and 24h (Cy5), and comparing their gene expression to untreated cells (Cy3). A signature of JAK signaling in ABC DLBCL was generated by treating HBL-1 cells with JAK inhibitor I (5 micromolar; Calbiochem) for 2h, 4h, 6h, and 8h (Cy5) and comparing their gene expression to vehicle-treated cells (DMSO, Cy3). RNA-Seq data not provided.

Project description:Constitutive activation of the anti-apoptotic NF-ÎºB signaling pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL) that is characterized by adverse survival. Recurrent oncogenic mutations are found in the scaffold protein CARMA1 (CARD11) that connects B-cell receptor (BCR) signaling to the canonical NF-ÎºB pathway. We asked how far additional downstream processes are activated and contribute to the oncogenic potential of DLBCL-derived CARMA1 mutants. To this end, we expressed oncogenic CARMA1 mutants in the NF-ÎºB negative DLBCL lymphoma cell line BJAB. By a proteomic approach we identified recruitment of Î²-Catenin and its destruction complex consisting of APC, AXIN1, CK1Î± and GSK3Î² to oncogenic CARMA1. Recruitment of the Î²-Catenin destruction complex was independent of CARMA1-BCL10-MALT1 (CBM) complex formation or constitutive NF-ÎºB activation and promoted the stabilization of Î²-Catenin. Elevated Î²-Catenin expression was detected in cell lines and biopsies from ABC DLBCL that rely on chronic BCR signaling. Increased Î²-Catenin amounts alone were not sufficient to induce classical WNT target gene signatures, but could augment TCF/LEF dependent transcriptional activation in response to WNT signaling. In conjunction with NF-ÎºB, Î²-Catenin enhanced expression of immune suppressive IL-10 and repressed anti-tumoral CCL3, indicating that Î²-Catenin may induce a favorable tumor microenvironment. Thus, parallel activation of NF-ÎºB and Î²-Catenin signaling by gain-of-function mutations in CARMA1 can augment WNT stimulation and is required for maintaining high expression of distinct NF-ÎºB target genes and can thereby trigger cell intrinsic and extrinsic processes that promote DLBCL lymphomagenesis. CARMA1 mutants were expressed in BJAB, an NF-ÎºB negative GCB DLBCL lymphoma cell line. Gene expression induced by GCB DLBCL derived CARMA1 L225LI mutant was compared with the empty vector (mock) control, CARMA1 WT, CARMA1 point mutant R35A and with CARMA1 double mutant R35A/L225LI.

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IkB-like protein NFKBIZ regulates NF-kB signaling and is critical for survival of ABC DLBCL (NFKBIZ inhibition)

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