Lower NF-kB Research Articles

Predictive Molecular Biomarkers for HPV-Associated Head and Neck Squamous Cell Carcinoma

<h3>Purpose/Objective(s)</h3> We hypothesized that molecular characteristics of HPV-associated head and neck squamous cell carcinoma (HPV+ HNSCC) will predict tumor behavior and response to therapy. Using TCGA, we previously correlated genetic defects in NF-kB regulators with outcome. Here, we validate this prognostic biomarker and identify a gene expression classifier that correlates with outcome in HPV+ HNSCC. <h3>Materials/Methods</h3> Total (ribosomal-depletion) RNA and targeted DNA sequencing was performed on an institutional cohort of HPV+ OPSCC tumors. Defects in NF-kB regulator genes: <i>TRAF3, CYLD, TRAF2, MYD88, NF-kBIA, TNFAIP3, TRAF6, BIRC2, BIRC3, MAP3K14</i> were identified and correlated with patient outcome. Expression data from <i>TRAF3</i> and <i>CYLD</i> mutant tumors was used to identify an initial gene set of differentially expressed genes. This initial set was optimized using machine learning to classify tumors based on RNA expression to NF-kB active and inactive sub-groups. The final gene set, termed the NF-kB activity classifier (NAC), was then correlated with patient outcome. <h3>Results</h3> Genomic defects in regulators of NF-kB that correlated with outcome in HPV+ HNSCC were initially identified using TCGA data. Herein, we find this association to be again observed in an independent patient cohort. Specifically, patients whose tumors harbored defects in the above listed genes had significantly better overall (p=0.03) and progression-free survival (p=0.03) compared to patients whose tumors were wild-type for these genes. Because we noted differential expression of many genes in tumors with and without defects in TRAF3 and CYLD, we also created a gene expression classifier as a complimentary method to distinguish these subsets. Gene set enrichment analysis demonstrated that the optimized classifier gene set was markedly enriched in NF-kB target genes, thereby was named the NF-kB Activity Classifier (NAC). Using the NAC, two HPV+ HNSCC patient populations were identified using TCGA data. Patients whose tumors had high NF-kB activity had significantly better recurrence-free survival compared to patients whose tumors had low NF-kB activity. <h3>Conclusion</h3> To improve quality of life for HPV+ HNSCC survivors, therapeutic deintensification is a goal of physicians treating HPV+ HNSCC. A major barrier has been determining appropriate patients whose treatment can be de-escalated without sacrificing the high cure rate associated with standard high-dose concurrent chemoradiation. Currently, TNM classification and smoking history are the only criteria available without pathologic analysis of resected tumors and nodes or induction chemotherapy. Here, using an independent cohort, we validated a prognostic biomarker based on defects in NF-kB pathway genes and created a new RNA based prognostic classifier that also correlates with outcome.

The effect of extract purple sweet drop on nuclear factor kappa B and amyloid deposits in D-galactose induced dementia in rats

Background. Alzheimer’s dementia (AD) as a neurodegenerative disease requires preventive measures to reduce its progression. Beta amyloid plaques induce chronic inflammation and apoptosis in AD. Anthocyanins in purple sweet potato are potential for prevention and therapy of AD. Aim. This study aimed to determine the role of anthocyanins in purple sweet potato extract to prevent inflammation so as to provide a neuroprotective effect on D-galactose-induced rats. Material and methods. We used a randomized posttest only control group design. 32 male Wistar rats according to eligibility criteria were randomized into control and treatment groups. The treatment group was given purple sweet potato extract at a dose of 200mg/kgBW every day for 70 days. Both groups on day 15 induced dementia using D-galactose. Anti-inflammatory activity was evaluated from the examination of NFkB levels by ELISA technique and neuroprotective effects by immunohistochemical examination of amyloid plaque deposits. Results. The mean NFkB of the treatment group (1417.55+255.82) was lower than the control group (1672.23+202.80) which was significant (p&lt;0.05) and the amyloid plaque deposits in the treatment group were thinner than the control group. Conclusions. Administration of purple sweet potato extract to D-galactose-induced rats caused lower NFkB levels and thinner amyloid plaque deposits. The research implication is that administration of purple sweet potato extract can prevent inflammation and provide neuroprotection effects in D-galactose-induced rats.

Effect of hesperetin on systemic inflammation and hepatic injury after blunt chest trauma in rats

ABSTRACTWe investigated the protective effect of hesperetin on hepatic damage after blunt chest trauma in rats using histological and biochemical methods. We used 18 adult male rats in three groups of six: control, chest trauma and chest trauma + hesperetin. Chest trauma was caused by dropping a metal cylinder onto the right hemithorax. Hesperetin, 100 mg/kg, was administered orally for 7 days. At the end of the seventh day, liver tissue samples were obtained. Serum tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), alanine aminotransferase (AST), aspartate transferase (ALT) and lactate dehydrogenase (LDH) enzyme activities were measured in blood samples taken from the heart. The general structure of liver tissue was investigated using hematoxylin and eosin staining. Nuclear factor kappa beta (Nf-κβ) expression in liver tissue was determined by the indirect immunohistochemical method. Apoptosis was determined using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method. Decreased TNF-α, AST and ALT enzyme activity, fewer histopathological changes and lower Nf-kB expression were observed in the hesperetin treated group compared to the chest trauma group. We also found reduced hepatic apoptosis in the chest trauma + hesperetin group compared to the chest trauma group. Hesperetine inhibits liver damage by reducing proinflammatory cytokines and by suppressing Nf-κβ activity in a blunt chest trauma model in rats.

Low Level Of NF-Kb Activity Is Associated With Higher Response Rate To Bortezomib-Based Induction Therapy In Patients With Newly Diagnosed Multiple Myeloma

PurposeActivation of canonical and noncanonical NF-kB pathways plays a key role in multiple myeloma (MM) pathogenesis. Recent studies have shown that constitutive activation of NF-kB pathways is present in ∼15-20 % of newly diagnosed MM patients. Bortezomib is a potent selective inhibitor of NF-kB activation; however its cytotoxicity is mainly due to inhibition of the noncanonical NF-kB pathway. We have previously shown that the achievement of very good partial response (VGPR) after induction therapy prior to frontline autologous stem cell transplantation (ASCT) was a favourable prognostic factor for progression-free survival (Moreau et al, Blood 2011;117:3041-3044). Our aim was to correlate NF-kB activity, reflected by a gene signature of well-known NF-kB targets, with the response rate achieved with a bortezomib-based induction regimen prior to high-dose therapy and ASCT performed as part of frontline treatment in patients with symptomatic multiple myeloma. Patients and methodsOne hundred and ninety-nine patients with symptomatic MM were enrolled in the prospective randomized IFM2007-02 trial comparing 4 cycles of bortezomib-dexamethasone (VD) induction therapy versus 4 cycles of the triplet combination bortezomib-thalidomide-dexamethasone (VTD) prior to ASCT. Among these 199 patients, 114 included in the present study were available for gene expression profile testing and therefore analysed for NF-kB activity. For each patient, the NF-kB(10) index, a reliable measure of NF-kB activity in MM tumor cells (Demchenko et al, Blood 2010; 115: 3541-3552) was calculated. NF-kB(10) index is based on a transcription signature of 10 genes: IL2RG, NFKB2,TNFAIP3, NFKBIE, NFKBIA, RELB, CD74, PLEK, MALT1 and WNT10. Gene expression signature was obtained from Affymetrix Exon1.0 normalized data. A high level of NF-kB activity was defined by the NF-kB(10) index found in a cohort of 20 MM patients with biallelic deletions, identified by using Affymetrix SNP6.0 data, that inactivate negative regulators (cIAP1/2) of NF-kB pathways. Response to induction therapy was evaluated according to the IMWG criteria. ResultsResponses to induction therapy are shown in Table 1. In this subgroup analysis, the CR plus VGPR rates were not statistically different in both arms of the trial: 24 / 54 (44%) in the VTD arm versus 21 / 60 (35%) in the VD arm, P = .35. We subsequently analyzed the correlation between NF-kB activity and response in the whole cohort of 114 patients regardless of induction treatment.Table 1Table patient's responseVD (n = 60)VTD (n = 54)CR1414VGPR710PR3023stable63progression34We found that the level of NF-kB activity, based NF-kB(10) index, tested as a continuous variable, was strongly correlated with the quality of response, i.e. VGPR or better (P = .007 Wilcoxon test). We also investigated the cut-off value of NF-kB(10) index that could impact the response rate. Since high NF-kB(10) index was found in MM cells with known NF-kB mutations, we calculated the NF-kB(10) index in a control cohort of MM patients deleted in cIAP1/2. Patients of IFM2007-02 trial with a NF-kB(10) index (< 70.5) lower than the index of the control cohort were assigned to the low NF-kB activity group (50/114, 44%); therefore the remaining cases (64/114, 56%) presented with a high NF-kB activity. We found that patients with a reduced NF-kB(10) index displayed a significantly higher response rate (27/50; 54% vs 18/64; 28%, P= .007), as shown in Figure 1, indicating that the vast majority of patients with high NF-kB were not able to achieve at least VGPR. [Display omitted] ConclusionOur results show that a low level NF-kB activity is associated with a higher response rate (>VGPR) to bortezomib-based induction regimen. Patients with low NF-kB(10) index represent 44% of the cohort studied. Since NK-kB activity is related to both canonical and noncanonical pathways, and knowing that bortezomib-induced cytotoxicity is mostly due to the inhibition of the noncanonical pathway only, our data strongly suggest that bortezomib should be combined with drug also targeting the canonical pathway in order to induce a high response rate in patients with increased NF-kB activity, as shown in preclinical studies (Fabre et al, Clin Cancer Res. 2012;18:4669-4681. Disclosures:Moreau:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau. Off Label Use: FRONTLINE TREATMENT WITH CARFIZOMIB. Attal:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau. Facon:Janssen and Celgene: Speakers Bureau; Millennium, Onyx, Novartis, BMS, Amgen: Membership on an entity's Board of Directors or advisory committees.

Impact of Global and Gene-Specific DNA Methylation Pattern in Relapsed Multiple Myeloma Patients Treated with Bortezomib

Abstract 132 Background:There is increasing evidence on the importance of epigenetic mechanisms such as DNA methylation and acetylation in the pathogenesis of multiple myeloma (MM). A global DNA hypomethylation pattern with selective genes hypermethylated has been described in myeloma plasma cells when compared with normal plasma cells. This fact could constitute a potential target for the use of demethylating agents. The response to bortezomib, a widely used agent against myeloma cells through proteasome inhibition, is particularly variable in patients with relapsed or refractory disease. We examined both, the global DNA methylation pattern and methylation state in 30 genes, in DNA from bone marrow cells and correlated our findings with response, progression (PFS) and overall-survival (OS) to bortezomib in patients with relapsed myeloma. Methods:Seventy-five patients (37M/38F; median age 65 years, range 29 to 80) with relapsed MM were treated from December 2002 to March 2010 with bortezomib-based regimens at our institution. Median follow-up for patients alive was 31 months (range 6 to 45). Genomic DNA was isolated from bone marrow slides with plasma cell infiltration at the time of relapse using a commercial kit (Qiagen). Global methylation was determined in all patients by ELISA (Epigentek), obtaining the percentage of 5-methylcytosine (5-mC) present in total DNA. CpG island DNA methylation profile of 30 genes was determined in 42 patients by a DNA methylation PCR system based on methylation sensitive and/or dependent restriction enzymes digestion (Qiagen). These genes were selected based on either their potential impact on prognosis in previous reports, or on the pathogenesis of MM, involving several cellular pathways such as innate immune response (CD40, EP300, MIF, CBP, TGFB1, TGFBR2), cytokine receptors (CXCR4, CXCL12, IL6R, IL17RA), transcription factors (NFKB1, NFKBIB, IRF4), cytokine stimulus response (SOCS3), apoptosis (TNFRSF13C, TNFRSF21, TNFRSF25, BCL2L11), tumor suppression (TP53, BRCA1, DAPK1, CDH1, RASD1), cellular cycle control (CCNB1, CCND1, CCNA2, CCNE1, CDKN2A, CDKN1A) and efflux transporter (ABCG2). Results:Overall response (OR) was achieved in 62% of the patients (complete remission 6.7%, partial response 44% and minor response 10.7%), while 9 (12%) and 20 (26.7%) showed no response (NR) or progressive disease (PD), respectively. The median PFS and OS after bortezomib therapy were 6 and 19.6 months, respectively. A low global methylation status was observed (median 4.68% of 5-mC, range 0.02 to 13.6) and patients with more than 3.95% of total DNA methylated achieved better OS than patients with more unmethylated DNA (median 30 versus 15 months) (p=0.004; Figure 1). Concerning methylation on specific-genes, a methylation status lower than 3.97% in CXCR4 was correlated with a longer PFS after bortezomib treatment (p=0.009; Figure 2). Clustering analysis with methylation status for these genes, showed that NFkB presented a differential profile according to response to bortezomib (p=0.037). A relative low methylation percentage (lower than 6.7%) in this gene was also associated with longer OS after bortezomib treatment (p=0.015; Figure 3). A positive correlation was observed with high methylation status in NFkB and other genes involved in the same cellular pathway (NFKBIB, EP300, CBP, CCNA2, CCNB1) (p<0.025). Moreover, a combination of highly methylated global genome and low NFkB methylation status defined a specific subset of patients with better prognosis (p=0.005) in terms of OS. Finally, a multivariate analysis including number of previous treatment lines, autologous stem-cell transplantation, previous exposure to bortezomib as well as global and NFkB methylation status showed that only the last two variables retained significance (p=0.035, OR=0.43 and p=0.028, OR=3.4, respectively). Conclusion:In our study, a low methylation grade in the overall DNA was observed. A relative high methylation status in the global genome and low in NFkB were associated with longer OS after bortezomib therapy in patients with relapsed or refractory myeloma. These results could be explained through the potential cell effect mediated by bortezomib in the NFkb pathway. Finally, a subgroup of patients with an ominous prognosis associated with DNA methylation at relapse in spite of bortezomib treatment was identified. [Display omitted] [Display omitted] [Display omitted] Disclosures:Fernández de Larrea:Novartis: Honoraria; Janssen: Honoraria; Celgene: Honoraria. Cibeira:Janssen: Honoraria; Celgene: Honoraria. Rosiñol:Janssen: Honoraria; Celgene: Honoraria. Blade:Janssen: Honoraria; Celgene: Honoraria.

Activation of Classical and Alternative Nuclear Factor-kappaB (NF-kB) Pathways in Diffuse Large B-Cell Lymphomas.

Activation of the NF-kB pathway is involved in many human neoplasms. In this study we examined the status of the NF-kB canonical (IkB, p50/p65) and non-canonical (p52, RelB) pathways in diffuse large B-cell lymphomas (DLBCL), which are a common and heterogeneous group of lymphoid malignancies. DLBCL have been divided into activated B-cell (ABC) like, and germinal center B-cell (GCB) like subgroups, which have been reported to have high and low NF-kB activity, respectively. However, the nature of the NF-kB complexes in this lymphoma entity has not been previously evaluated. Therefore we performed Western blotting, electrophoretic mobility shift assays (EMSA) and real time quantitative RT-PCRs on nuclear and cytoplasmic protein and total RNA extracts in 20 primary tumor samples and 9 different DLBCL cell lines. In the cell lines, presence of NF-kB proteins in nuclear extracts correlated with expression of NF-kB target genes (CCR7, IkBa, CCND2, BCL-2, IRF4) as determined by RT-PCR. Therefore, these could be assigned into GCB and ABC-like categories. In primary DLBCLs, EMSA showed NF-kB binding in all but one case. The same cases (19/20) had high p52 in the nucleus indicating activation of the alternative pathway. TNF family ligand BAFF was also found to be expressed in all primary samples and most cell lines. The classical pathway, as determined by nuclear p50, was also present in these cases. Levels of p65 and RelB expression were variable, but did not correlate with the mRNA expression of NF-kB target genes. In conclusion, while DLBCL cell lines may be divided into two distinct categories, the primary samples represented a spectrum of NF-kB target gene activity, while levels of NF-kB expression were high. BAFF expression and activation of the alternative pathway may be important in the pathogenesis of DLBCL.