scholarly journals NF-κB p50 activation associated with immune dysregulation confers poorer survival for diffuse large B-cell lymphoma patients with wild-type p53

2017 ◽  
Vol 30 (6) ◽  
pp. 854-876 ◽  
Author(s):  
Qingqing Cai ◽  
Meifeng Tu ◽  
Zijun Y Xu-Monette ◽  
Ruifang Sun ◽  
Ganiraju C Manyam ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Immune Dysregulation ◽  
Wild Type ◽  
Large B Cell Lymphoma ◽  
Wild Type P53 ◽  
Large B Cell

scholarly journals MicroRNA-181a Inhibits Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Progression by Repressing CARD11

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Danxia Zhu ◽  
Cheng Fang ◽  
Wenting He ◽  
Chen Wu ◽  
Xiaodong Li ◽  
...  
Keyword(s):  
B Cell ◽  
Binding Site ◽  
Cell Lines ◽  
Expression Vector ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
Protein Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

We investigated the role of miR-181a in diffuse large B-cell lymphoma (DLBCL) and its potential target genes. miR-181a levels were lower in activated B-cell- (ABC-) like DLBCL cells than that in germinal center B-cell- (GCB-) like DLBCL cells. Overexpression of miR-181a in ABC-like DLBCL cell lines (OCI-LY10 and U2932) resulted in G0/G1 cell cycle arrest, increased apoptosis, and decreased invasiveness. miRNA target prediction programs (miRanda, TargetScan, and miRDB) identified caspase recruitment domain-containing protein 11 (CARD11) as a putative miR-181a target. CARD11 mRNA and protein levels were higher in the ABC-like DLBCL than that in GCB-like DLBCL. Moreover, CARD11 mRNA and protein levels were downregulated in the OCI-LY10 and U2932 cell lines overexpressing miR-181a. Dual luciferase reporter assays confirmed the miR-181a binding site in the CARD11 3′UTR region. OCI-LY10 and U2932 cells transfected with a CARD11 expression vector encoding miR-181a with a mutated binding site showed higher CARD11 protein levels, cell viability, G2/M phase cells, and invasiveness compared to those transfected with a wild-type CARD11 expression vector. Nude mice xenografted with OCI-LY10 cells with overexpressed wild-type miR-181a generated smaller tumors compared to those with overexpressed mutated binding site of CARD11 3′UTR and miR-181a. These results indicate that miR-181a inhibits ABC-like DLBCL by repressing CARD11.

scholarly journals Somatic PRAME Deletions Are Associated with Decreased Immunogenicity, Apoptosis Resistance and Poor Outcomes in Diffuse Large B-Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 667-667
Author(s):  
Katsuyoshi Takata ◽  
Daisuke Ennishi ◽  
Ali Bashashati ◽  
Saeed Saberi ◽  
Elena Viganò ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
Memory T Cell ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background: The current standard of care in diffuse large B-cell lymphoma (DLBCL) consists of chemotherapy and therapeutic monoclonal antibodies that have significantly improved patient outcomes over the past 15 years. However, a large proportion of patients suffer from refractory or relapsed disease. Therefore, the development of new therapeutic strategies for this subgroup of patients, who are threatened by a high chance of disease-related death, represents an important unmet clinical need. Methods: We enrolled into our study 347 de novo DLBCL patients uniformly treated with R-CHOP from the BC Cancer population-based cohort between September 2000 and January 2012. RNAseq and high-resolution copy number analysis were performed and correlated with clinical outcome data and tumor microenvironment composition. We also performed functional studies to investigate PRAME-mediated memory T-cell responses and gene expression changes. Results: We discovered novel, highly focal deletions of 22q11.22, including the PRAME gene in 13% (44/338) of the cases. The deletions cluster in a narrow chromosomal region that includes a very small number of genes (VpreB1, ZNF280A/B, PRAME, GGTLC2, miR-650). Of clinical importance, 22q11.22 deletions were found significantly more frequently in germinal centre B-cell-like (GCB) type DLBCL (17% (31/180) vs. activated B-cell-like (ABC) type: 8% (8/98), P < 0.01), and were also significantly associated with worse outcome, which was specifically observed in GCB-DLBCL (5-year disease specific survival, non-PRAME-deleted: 84.5% vs. PRAME-deleted: 67.2%, P = 0.026). Homozygous deletions were more strongly associated with poor outcome than heterozygous deletions. Interestingly, 90% of PRAME-deleted cases were Ig-lambda restricted (P < 0.001). PRAME is a prominent member of the cancer testis antigen (CTA) family of proteins that are expressed in various types of cancers, but not in normal tissues, including normal mature B-cells, apart from male germinal cells. Due to the cancer-specific expression of CTAs, these molecules are considered promising targets for cancer immunotherapy using cytotoxic T-cells and tumor vaccination approaches. To determine the association with tumor microenvironment composition, we analyzed CD4/CD8 flow cytometry data from DLBCL patient samples. The numbers of CD4 and CD8-positive T cells were significantly lower in PRAME-deleted cases compared to wild type (CD4: P < 0.001, CD8: P = 0.013). Notably, RNAseq analysis revealed that the HLA-A*0201 genotype was seen significantly more often in PRAME deleted cases (PRAME wt: 2.5% vs. PRAME deleted: 10.8%, P = 0.005). In order to functionally characterize its interaction with the immune microenvironment, we utilized enzyme-linked immunoSpot (ELISPOT) assays to investigate memory T-cell reactions of patient-derived T cells to PRAME antigens using patient-derived peripheral blood mononuclear cells (PBMC) and measured IFN-g production (7 control healthy donors, 4 PRAME-deleted and 4-wild type patients). While T cells from PRAME-replete patients had no reaction to PRAME antigens, PRAME-deleted patient-derived T-cells had significant reactions to 4 independent PRAME peptides. These data suggest that PRAME-deleted tumor cells can escape from cytotoxic T-cell attack to gain growth advantage. Next, we performed PRAME knock-out (KO) experiments using CRISPR/Cas9 genome editing to clarify the cell autonomous effects of PRAME deletions. Using 2 different cell lines (Karpas422 and SUDHL-4), we found TNFSF10 (TRAIL) expression was significantly down-regulated in homozygous PRAME-KO cell lines compared to wild type. The soluble form of TRAIL (sTRAIL) was also reduced, as measured with enzyme-linked immunosorbent assays. These results suggest that PRAME downregulated cells may contribute to cell survival via TRAIL and sTRAIL reduction. Conclusion: We identified recurrent PRAME deletions and characterized their clinical and functional role in DLBCL. Our findings contribute to the understanding of cell-autonomous and extrinsic roles of PRAME deletions in lymphomagenesis and may lead to the discovery of new therapeutic avenues to simultaneously treat the tumor and the host. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Janssen: Research Funding; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria. Steidl:Tioma: Research Funding; Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

Poor Survival Predicted by MDM2 Oncoprotein Expression in Diffuse Large B-Cell Lymphoma (DLBCL) with Wild-Type TP53 Gene

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5269-5269 ◽  
Author(s):  
Gabrielle L. Rocque ◽  
Michael B. Møller ◽  
Gisele W. B. Colleoni ◽  
Margarita Sánchez-Beato ◽  
Tina M. Green ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Tp53 Gene ◽  
Wild Type ◽  
P53 Pathway ◽  
Mdm2 Protein ◽  
Large B Cell Lymphoma ◽  
Mdm2 Expression ◽  
Large B Cell

Abstract The p53 pathway is known to play a role in pathogenesis and prognosis of human malignancies including diffuse large B-cell lymphoma (DLBCL). The MDM2 protein has been shown to regulate p53 function and stability via a multi-factorial negative feedback loop. MDM2 is involved in transport of p53 out of the nucleus and its subsequent degradation. Additionally, MDM2 binds p53 protein and inhibits its function as a transcription factor. As an integral part of the regulation of the p53 pathway, we hypothesize MDM2 expression contributes to the pathogenesis of DLBCL and affects survival. The purpose of this study is to investigate the potential role of the MDM2 protein in DLBCL by correlating expression of MDM2 with p53 expression, TP53 mutation status and clinical outcome. Immunostains for MDM2 and TP53 gene-product proteins were performed in 133 cases from 6 medical centers. A positive immunostain was defined as nuclear staining in 10% or more of the tumor cells. The TP53 gene was analyzed for mutations with PCR-based and sequencing methods. Patients were treated with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) or CHOP-like regimen. The Kaplan-Meier method was used for survival analysis. MDM2 immunostains were positive in 64 of 133 cases of DLBCL (48%). There was a trend toward poor overall survival (OS) with MDM2 expression in the entire group of patient with DLBCL (p=0.32). Twelve of 24 cases (50%) with TP53 mutations demonstrated MDM2 expression. Within this TP53 mutated group, no significant difference in OS was observed between MDM2-positive and MDM2-negative cases (p=0.32). Of patients with wild-type (WT) TP53, 51 of 107 cases (48%) expressed MDM2. In this subset of patients, the MDM2-positive phenotype predicted for poor 5-year OS (38% vs 67%, p=0.002), and a significantly shorter median disease-free survival (2.3 years vs. &gt;5.0 years; p=0.013). The complete remission rate was 57% in MDM2-positive cases compared to 73% in MDM2-negative cases in the WT-TP53 group (p=0.08). Multivariate analysis confirmed that MDM2 expression was an independent predictor of poor OS in patient with DLBCL with a WT-TP53 gene (HR 2.0, 95% CI 1.15–3.56; p=0.015). This study demonstrates the negative survival impact of MDM2 expression in patients with wild-type TP53, suggesting that MDM2 provides an alternative mechanism of p53 pathway inactivation in DLBCL cases with a WT-TP53 gene.

Nuclear Import of Janus Kinase 1 Is Mediated By Multiple Alpha Importins and Is Required for the Survival of Diffuse Large B-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 780-780
Author(s):  
Lixin Rui
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
B Cell ◽  
Nuclear Transfer ◽  
Cell Lymphoma ◽  
Nuclear Transport ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Janus kinases (JAKs) are non-receptor tyrosine kinases that are generally found bound to cytokine receptors. JAKs have well established roles in activating signal transducers and activators of transcription (STATs) in response to cytokine stimulation in the cytoplasm. However, the epigenetic regulation of gene transcription by nuclear JAK2 through phosphorylation of tyrosine 41 on the histone protein H3 has recently been discovered. Our very recent work demonstrated that this histone targeting mechanism applies to another member of the JAK family, JAK1, which is activated by the autocrine cytokines IL6 and IL10 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). Genome-wide mapping of the H3Y41p histone mark revealed 2956 potential JAK1 target genes, 91% of which do not have a STAT motif in their promoter region. Here we investigated the JAK1 nuclear transfer mechanism in ABC DLBCL. Using cell fractionation and immunofluorescence assays, we detected a nuclear pool of JAK1 in both HEK 293T cells and ABC DLBCL cell lines. In searching for a putative nuclear localization signal (NLS) of JAK1, we identified a basic amino acid enriched sequence (KRKK…KHKK, starting from amino acid 342) that was reminiscent of a classical bipartite NLS. Immunofluorescence with GFP-tagged JAK1 truncations and full length JAK1 with mutations in putative NLS revealed that the first group of amino acids (KRKK) was the NLS that was responsible for the nuclear transport of JAK1. Our results, however, indicate that the active transport of JAK1 into the nucleus is independent of its activation status, since nucleocytoplasmic distribution did not change either in HEK293T cells when constitutively activated JAK1 was expressed or in ABC DLBCL cells with IL-6 stimulation or in GCB DLBCL cells that lack cytokine signaling. To identify potential importins that recognize the NLS of JAK1 and subsequently transport JAK1 into the nucleus, we used immunoprecipitation and GST pull down assays. We found that importin a4, a5 and a7 (encoded by KPNA3, KPNA1, and KPNA6 respectively) physically interacted with the NLS of JAK1 and these interactions were abolished when the 4 amino acids in the NLS were mutated to alanine, suggesting these alpha importins recognize the NLS of JAK1 (KRKK) and mediate the nuclear transport of JAK1. To test whether the NLS-mediated nuclear transfer that allows JAK1 to function in the nucleus is important for the fitness of ABC DLBCL cells, we performed JAK1 knockdown and rescue experiments in TMD8 and OCI-Ly10 cells, in which endogenous JAK1 was knocked down by an shRNA and an shRNA-resistant cDNA of either wild type or NLS-defective JAK1 was constantly expressed. The results revealed that knockdown of JAK1 led to reduced viable cells in the culture, suggesting that JAK1 expression is required for cancer cell survival. As expected, overexpression of wild type JAK1 completely reversed the toxicity due to knockdown of endogenous JAK1. However, overexpression of the NLS-defective JAK1 did not rescue the toxic effects of JAK1 shRNA. Thus, the findings suggest that nuclear transport of JAK1 is an essential process to maintain the survival of ABC DLBCL, and targeting the JAK1 NLS might be a potential targeted therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

scholarly journals SIRT3, a metabolic target linked to ataxia-telangiectasia mutated (ATM) gene deficiency in diffuse large B-cell lymphoma

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kavita Bhalla ◽  
Sausan Jaber ◽  
Kayla Reagan ◽  
Arielle Hamburg ◽  
Karen F. Underwood ◽  
...  
Keyword(s):  
B Cell ◽  
Ataxia Telangiectasia ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
Ataxia Telangiectasia Mutated ◽  
Large B Cell Lymphoma ◽  
Atm Gene ◽  
Sirt3 Expression ◽  
Large B Cell

AbstractInactivation of Ataxia-telangiectasia mutated (ATM) gene results in an increased risk to develop cancer. We show that ATM deficiency in diffuse large B-cell lymphoma (DLBCL) significantly induce mitochondrial deacetylase sirtuin-3 (SIRT3) activity, disrupted mitochondrial structure, decreased mitochondrial respiration, and compromised TCA flux compared with DLBCL cells expressing wild type (WT)-ATM. This corresponded to enrichment of glutamate receptor and glutamine pathways in ATM deficient background compared to WT-ATM DLBCL cells. ATM−/− DLBCL cells have decreased apoptosis in contrast to radiosensitive non-cancerous A-T cells. In vivo studies using gain and loss of SIRT3 expression showed that SIRT3 promotes growth of ATM CRISPR knockout DLBCL xenografts compared to wild-type ATM control xenografts. Importantly, screening of DLBCL patient samples identified SIRT3 as a putative therapeutic target, and validated an inverse relationship between ATM and SIRT3 expression. Our data predicts SIRT3 as an important therapeutic target for DLBCL patients with ATM null phenotype.

scholarly journals Molecular and Genetic Characterization of MHC Deficiency Identifies EZH2 As a Therapeutic Target for Restoring MHC Expression in Diffuse Large B-Cell Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1560-1560
Author(s):  
Daisuke Ennishi ◽  
Katsuyoshi Takata ◽  
Wendy Béguelin ◽  
Gerben Duns ◽  
Anja Mottok ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
Mhc I ◽  
Mhc Ii ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Introduction: Among the tumor immune escape mechanisms described to date, alterations in the expression of major histocompatibility complex (MHC) molecules play a crucial role in the development of diffuse large B-cell lymphoma (DLBCL). Although the frequency of loss of MHC expression differs between ABC- and GCB-DLBCL cell of origin (COO) subtypes, distinct genetic alterations and molecular features that affect MHC expression and the composition of immune cells in the tumor microenvironment remain ill-defined. Here, we aimed to uncover the biologic and genomic basis underlying acquired loss of MHC expression. Method: We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL and uniformly treated with R-CHOP in British Columbia. We performed targeted resequencing, SNP6.0 array and RNAseq for genetic analyses. Immunohistochemical (IHC) staining of MHC-I and -II was performed on tissue microarrays (n=332). COO was assigned by the Lymph2Cx assay in 323 cases (183 GCB, 104 ABC and 36 unclassifiable). Immune cell composition was assessed by IHC, flow cytometry and gene expression profiling (GEP)-based deconvolution of cellular signatures. To experimentally confirm decreased MHC expression induced by EZH2 mutation, we measured surface MHC-I and -II expression on tumor B cells using EZH2Y641/BCL2 mouse model which was previously established (Beguelin et al, Cancer Cell 2013). We also treated human DLBCL cells harboring EZH2 mutation and wild type using EZH2 inhibitor (EPZ-6438), and evaluated their surface MHC-I and -II expression. Results: Loss of MHC-I and -II expression was observed in 43% and 28% of DLBCL cases, respectively. MHC-II loss of expression was significantly associated with the reduction of tumor-infiltrating lymphocytes (TILs), especially CD4 positive T-cells (FOXP3+ cells, PD-1+ cells, and CD4+ naïve and memory T-cells), and cytolytic activity (GZMB and PRF1 mRNA expression) in GCB-DLBCL (all; p<0.001), but not in ABC-DLBCL. MHC-II-negativity was associated with unfavorable prognosis only in GCB-DLBCL (5-year time-to-progression; 59% vs 79%, p=0.007), whereas there was no prognostic impact of MHC-I expression in either subtype, suggesting a link between loss of MHC-II expression and reduced immune surveillance leading to poor prognosis, specifically in GCB-DLBCL. We next performed GEP using RNAseq separately in each COO subtype. Interestingly, only four genes (HLA-DMA, DRA, DPA1 and CD74) were differentially expressed according to MHC-II expression (FDR<0.001) in ABC-DLBCL. By contrast, a total of 641 genes were differentially expressed in GCB-DLBCL. Of importance, a dark zone (DZ) B-cell signature was strongly enriched in MHC-II-negative GCB-DLBCL cases (FDR<0.001), suggesting that MHC-II deficiency defines the tumor originated from DZ of the germinal center. Correlative genetic analysis revealed that, as expected, mutations of CIITA and RFXAP were detected more frequently in MHC-II-negative GCB-DLBCL (p=0.01 and 0.003, respectively). Strikingly, CD83 mutations, which elevate and stabilize MHC-II expression in centrocytes of the light zone (LZ), were significantly enriched in MHC-II positive GCB-DLBCL (p= 0.008), suggesting that these mutations affecting the antigen presentation machinery are selectively acquired in GCB-DLBCL tumors to further reduce and increase the surface MHC-II expression. Genetic analysis also highlighted that EZH2 mutations were most significantly enriched in MHC-II-negative as well as MHC-I-negative GCB-DLBCL cases (both, p<0.001). Indeed, 77% of EZH2 mutated cases demonstrated loss of either MHC-I and/or MHC-II expression on the tumor cells. Notably, we found significantly lower MHC-I and MHC-II expression in high-grade lymphomas of EZH2 mutant Vav-BCL2 transgenic mice compared to EZH2 wildtype control tumors. Furthermore, of potential clinical relevance, in-vitro EZH2 inhibition significantly restored MHC-I and MHC-II gene expression as well as protein expression in EZH2-mutated human DLBCL cells, but not EZH2 wild type tumor cells. Conclusion: Our findings provide important implications for understanding the cancer biology underlying acquired loss of MHC expression. The restoration of MHC expression by EZH2 inhibitors suggests a novel approach of epigenetically enhancing tumor recognition and eradication in combination with immune therapies. Disclosures Sehn: Abbvie: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria. Connors:Roche Canada: Research Funding; Takeda: Research Funding; Merck: Research Funding; F Hoffmann-La Roche: Research Funding; Cephalon: Research Funding; Seattle Genetics: Honoraria, Research Funding; Amgen: Research Funding; Bayer Healthcare: Research Funding; Bristol Myers-Squibb: Research Funding; Lilly: Research Funding; NanoString Technologies: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Janssen: Research Funding; Genentech: Research Funding. Gascoyne:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Roche: Research Funding; Janssen: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria. Steidl:Juno Therapeutics: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Nanostring: Patents & Royalties: patent holding; Bristol-Myers Squibb: Research Funding; Tioma: Research Funding.

scholarly journals KLHL14 Is a Tumor Suppressor in Diffuse Large B-Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2766-2766
Author(s):  
Jaewoo Choi ◽  
James D Phelan ◽  
Ryan M. Young ◽  
Thomas Oellerich ◽  
Da Wei Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Somatic Mutations ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Loss Of Function ◽  
Wild Type ◽  
Large B Cell Lymphoma ◽  
Inactivating Mutations ◽  
Large B Cell

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer of aberrant B-lymphocytes. Although a portion of DLBCL is curable with standard immunochemotherapy, patients who fail this treatment have a poor prognosis. Recently, cancer genomics has paved the way for better understanding of the genetic basis of lymphoma pathogenesis. Characterization of point mutations and structural alterations has uncovered novel molecular targets for lymphoma therapy and provided a comprehensive view of lymphoma development. By performing multiplatform genomic analysis of DLBCL biopsy samples, we have identified KLHL14 as a recurrent target of somatic mutations in activated B-cell-like (ABC) DLBCL biopsies (10.8% of patients). KLHL14 contains a BTB (broad complex, tramtrack, and bric a brac) domain that can potentially mediate dimerization and binding to Cullin3 (CUL3)-a essential scaffold component of the Cullin-RING-based E3 ubiquitin ligase complexes. KLHL14 also contains kelch repeats that can form a B-propeller tertiary structure that can serve as a substrate-binding domain. KLHL14 is highly expressed in B-cells but is found at low levels in non-immune tissues. Deficiency of KLHL14 in mice leads to embryonic lethality while KLHL14 heterozygous mice show reduction of B-1a cells, suggesting a role for KLHL14 in B-cell homeostasis. Importantly, KLHL14 mutations are highly enriched in tumors belonging to the recently defined MCD (MYD88L265P/CD79B mutation) genetic subtype of DLBCL, the subset of ABC DLBCLs. Somatic mutations primarily localize to the N-terminus of the protein in the BTB domain and BACK (BTB and C-terminal Kelch) domain. However, the impact of these mutations as well as the molecular function of KLHL14 is largely unknown. To investigate the biological effect of KLHL14 loss of function, we used an inducible CRISPR/Cas9 system to delete KLHL14 in ABC DLBCL cell lines and monitored cell growth. Ablation of KLHL14 resulted in an increase in cell proliferation and survival, supporting a role for KLHL14 as a tumor suppressor. Next, we performed a multiplatform -omic analysis (proteomics, phosphoproteomics, ubiquitinomics, high-throughput sequencing) to explore the signaling networks and interactome of KLHL14. Whereas ectopic expression of wild-type KLHL14 altered the dynamics of tyrosine phosphorylation and ubiquitylation events in ABC DLBCL lines, KLHL14 lymphoma-associated mutant alleles had little if any effect, suggesting that they are loss-of-function variants. Gene expression profiling by RNA-sequencing revealed that KLHL14-inactivated cells have a higher NF-kB target gene expression than wild-type cells. Thus, tumor-associated inactivating mutations of KLHL14 depend on a subset of essential NF-kB-related oncoproteins for their survival and this might contribute to the proliferative advantage of DLBCL. In summary, we have uncovered a tumor suppressive function of KLHL14 and found that KLHL14 mutants promote ABC DLBCL survival by increasing NF-kB activity. These findings suggest that tumors with KLHL14 inactivating mutations may serve as a marker of resistance to anti-NF-kB treatment and provide the basis for treating MCD subtype patients with downstream NF-kB pathway inhibitors in the clinical settings. Disclosures Staudt: Nanostring: Patents & Royalties.

ZLD1122, a novel EZH2 and EZH1 small molecular inhibitor, blocks H3K27 methylation and diffuse large B cell lymphoma cell growth

RSC Advances ◽  
2016 ◽  
Vol 6 (34) ◽  
pp. 28512-28521 ◽  
Author(s):  
Tiantao Gao ◽  
Lidan Zhang ◽  
Yongxia Zhu ◽  
Xuejiao Song ◽  
Qiang Feng ◽  
...  
Keyword(s):  
Cell Growth ◽  
B Cell ◽  
Small Molecule ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Wild Type ◽  
H3k27 Methylation ◽  
Large B Cell Lymphoma ◽  
Molecule Inhibitor ◽  
Large B Cell

Here, we reported a novel, selective, small-molecule inhibitor of EZH2 and EZH1 synthesized by us, ZLD1122, which inhibited both EZH1 and wild type and mutant EZH2 activities with nanomolar potency.

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