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TRAF3 loss-of-function Reveals the Non-Canonical NF-κB Pathway as a Therapeutic Target in Diffuse Large B-cell Lymphoma

ABSTRACT: Treatment of diffuse large B-cell lymphoma (DLBCL) remains challenging due to extensive molecular, clinical, and pathological heterogeneity. Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3, a negative regulator of NF-κB signaling, in a cohort of uniformly-treated de novo DLBCL (24/324 cases). Integrative analysis uncovered a correlation between TRAF3 copy number loss and TRAF3 reduced expression. CRISPR-mediated TRAF3 loss-of-function (LOF) in DLBCL cell lines enhanced non-canonical NF-κB (NC NF-κB) signaling, rendering cells sensitive to shRNA-induced knockdown of the central NC NF-κB kinase, NIK. NIK pharmacological inhibitors differentially impaired proliferation, and induced apoptosis of TRAF3 LOF cells, further suggesting an acquired onco-addiction to NC NF-κB. Beyond these cell-intrinsic effects, co-culturing of TRAF3 LOF DLBCL cells with primary human CD8+ T-cells revealed an impairment in effector marker induction (Granzyme B, IFNγ) and proliferation in the latter. Accordingly, a reduction in T-cell infiltrates was observed in the microenvironment of TRAF3-low expressing primary DLBCL tumor samples. Neutralization of IL10 produced by TRAF3 LOF cells restored and enhanced GZMB and IFNγ expression in co-cultured CD8+ T-cells. Our findings demonstrate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, favoring pro-oncogenic cell-intrinsic effects and immune-evasive mechanisms, and highlight NIK as a therapeutic target in defined subset of DLBCL.

ORGANISM(S): Homo sapiens

PROVIDER: GSE218750 | GEO | 2024/04/11

REPOSITORIES: GEO

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Project description:The NF-κB pathway is a master regulator of inflammatory processes and is implicated in insulin resistance and pancreatic beta cell dysfunction in the metabolic syndrome. While canonical NF-κB signaling is well studied, there is little information on the divergent non-canonical NF-κB pathway in the context of pancreatic islet dysfunction in diabetes. Here, we demonstrate that pharmacological activation of the non-canonical NF-κB inducing kinase (NIK) disrupts glucose homeostasis in zebrafish in vivo. Further, we identify NIK as a critical negative regulator of beta cell function as pharmacological NIK activation results in impaired glucose-stimulated insulin secretion in mouse and human islets. NIK levels are elevated in pancreatic islets isolated from diet-induced obese (DIO) mice, which exhibit increased processing of non-canonical NF-κB components p100 to p52, and accumulation of RelB. Tumor necrosis factor α (TNFα) and receptor activator of NF-κB ligand (RANKL), two ligands associated with diabetes, induce NIK in islets. Mice with constitutive beta cell intrinsic NIK activation present impaired insulin secretion with DIO. NIK activation triggers the non-canonical NF-κB transcriptional network to induce genes identified in human type 2 diabetes genome-wide association studies linked to beta cell failure. These studies reveal that NIK contributes a central mechanism for beta cell failure in diet-induced obesity. We identify a role for Nuclear Factor inducing κB (NIK) in pancreatic beta cell failure. NIK activation disrupts glucose homeostasis in zebrafish in vivo and impairs glucose-stimulated insulin secretion in mouse and human islets in vitro. NIK activation also perturbs beta cell insulin secretion in a diet-induced obesity mouse model. These studies reveal that NIK contributes a central mechanism for beta cell failure in obesity. To uncover the role of NIK in pancreatic beta cells, we performed a gene expression microarray analysis comparing pancreatic islets with constitutive beta cell intrinsicNIK activation from the 16 week old mice (beta cell specific TRAF2 and TRAF2 knockout mice) to their controls (n=3 per group).

Project description:Proper regulation of nuclear factor κB (NF-κB) transcriptional activity is required for normal lymphocyte function, and deregulated NF-κB signaling can facilitate lymphomagenesis. We demonstrate that the API2-MALT1 fusion oncoprotein created by the recurrent t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma induces proteolytic cleavage of NF-κB–inducing kinase (NIK) at arginine 325. NIK cleavage requires the concerted actions of both fusion partners and generates a C-terminal NIK fragment that retains kinase activity and is resistant to proteasomal degradation. The resulting deregulated NIK activity is associated with constitutive noncanonical NF-κB signaling, enhanced B cell adhesion, and apoptosis resistance. Our study reveals the gain-of-function proteolytic activity of a fusion oncoprotein and highlights the importance of the noncanonical NF-κB pathway in B lymphoproliferative disease. This study compares nine t(11;18)-positive MALT lymphomas (8 from the stomach and 1 from lung) and eight translocation negative MALT lymphomas (all from the stomach) using gene set enrichment analysis (GSEA). All cases were subjected to Affymetrix U133A and U133B microarray analysis. The cases used in this study are the same cases used for the study by Hamoudi et al. (2010) entitled "Differential expression of NF-kB target genes in MALT lymphoma with and without chromosome translocation: insights into molecular mechanism" with GEO reference number: GSE18736 and PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/20520640 All cases were subjected to non-specific filtering to eliminate non-variant probes, then the U133A and U133B probes were collapsed and the collapsed set was subjected to GSEA using the NF-kB target gene set as described in Hamoudi et al. (2010) study mentioned above. The 34 samples in this study are identical to the ones done in the previous series except that the gene set enrichment was done on just those 34 samples and not the complete set.

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TRAF3 loss-of-function Reveals the Non-Canonical NF-κB Pathway as a Therapeutic Target in Diffuse Large B-cell Lymphoma

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