CDK1 is up-regulated by temozolomide in an NF-κB dependent manner in glioblastoma.

The alkylating agent, temozolomide (TMZ), is the most commonly used chemotherapeutic for the treatment of glioblastoma (GBM). The anti-glioma effect of TMZ involves a complex response that includes G2-M cell cycle arrest and cyclin-dependent kinase 1 (CDK1) activation. While CDK1 phosphorylation is a well-described consequence of TMZ treatment, we find that TMZ also robustly induces CDK1 expression. Analysis of this pathway demonstrates that CDK1 is regulated by NF-κB via a putative κB-site in its proximal promoter. CDK1 was induced in a manner dependent on mature p50 and the atypical inhibitor κB protein, BCL-3. Treatment with TMZ induced binding of NF-κB to the κB-site as assessed by gel shift analysis and chromatin immunoprecipitation. Examination of a CDK1 promoter-reporter demonstrated the functional relevance of the κB-site and underlined the requirement of p50 and BCL-3 for activation. Targeted knockdown of CDK1 or chemical inhibition with the selective CDK1 inhibitor, RO-3306, potentiated the cytotoxic effect of TMZ. These results identify CDK1 as an NF-κB target gene regulated by p50 and BCL-3 and suggest that targeting CDK1 may be a strategy to improve the efficacy of TMZ against GBM.

p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability.

p50, the mature product of NFKB1, is constitutively produced from its precursor, p105. Here, we identify BARD1 as a p50-interacting factor. p50 directly associates with the BARD1 BRCT domains via a C-terminal phospho-serine motif. This interaction is induced by ATR and results in mono-ubiquitination of p50 by the BARD1/BRCA1 complex. During the cell cycle, p50 is mono-ubiquitinated in S phase and loss of this post-translational modification increases S phase progression and chromosomal breakage. Genome-wide studies reveal a substantial decrease in p50 chromatin enrichment in S phase and Cycln E is identified as a factor regulated by p50 during the G1 to S transition. Functionally, interaction with BARD1 promotes p50 protein stability and consistent with this, in human cancer specimens, low nuclear BARD1 protein strongly correlates with low nuclear p50. These data indicate that p50 mono-ubiquitination by BARD1/BRCA1 during the cell cycle regulates S phase progression to maintain genome integrity.

DDX39B interacts with the pattern recognition receptor pathway to inhibit NF-κB and sensitize to alkylating chemotherapy.

BACKGROUND: Nuclear factor-κB (NF-κB) plays a prominent role in promoting inflammation and resistance to DNA damaging therapy. We searched for proteins that modulate the NF-κB response as a prerequisite to identifying novel factors that affect sensitivity to DNA damaging chemotherapy. RESULTS: Using streptavidin-agarose pull-down, we identified the DExD/H-box RNA helicase, DDX39B, as a factor that differentially interacts with κB DNA probes. Subsequently, using both RNA interference and CRISPR/Cas9 technology, we demonstrated that DDX39B inhibits NF-κB activity by a general mechanism involving inhibition of p65 phosphorylation. Mechanistically, DDX39B mediates this effect by interacting with the pattern recognition receptor (PRR), LGP2, a pathway that required the cellular response to cytoplasmic double-stranded RNA (dsRNA). From a functional standpoint, loss of DDX39B promoted resistance to alkylating chemotherapy in glioblastoma cells. Further examination of DDX39B demonstrated that its protein abundance was regulated by site-specific sumoylation that promoted its poly-ubiquitination and degradation. These post-translational modifications required the presence of the SUMO E3 ligase, PIASx-ß. Finally, genome-wide analysis demonstrated that despite the link to the PRR system, DDX39B did not generally inhibit interferon-stimulated gene expression, but rather acted to attenuate expression of factors associated with the extracellular matrix, cellular migration, and angiogenesis. CONCLUSIONS: These results identify DDX39B, a factor with known functions in mRNA splicing and nuclear export, as an RNA-binding protein that blocks a subset of the inflammatory response. While these findings identify a pathway by which DDX39B promotes sensitization to DNA damaging therapy, the data also reveal a mechanism by which this helicase may act to mitigate autoimmune disease.

Temozolomide Treatment Induces lncRNA MALAT1 in an NF-κB and p53 Codependent Manner in Glioblastoma.

Alkylating chemotherapy is a central component of the management of glioblastoma (GBM). Among the factors that regulate the response to alkylation damage, NF-κB acts to both promote and block cytotoxicity. In this study, we used genome-wide expression analysis in U87 GBM to identify NF-κB-dependent factors altered in response to temozolomide and found the long noncoding RNA (lncRNA) MALAT1 as one of the most significantly upregulated. In addition, we demonstrated that MALAT1 expression was coregulated by p50 (p105) and p53 via novel κB- and p53-binding sites in the proximal MALAT1 coding region. Temozolomide treatment inhibited p50 recruitment to its cognate element as a function of Ser329 phosphorylation while concomitantly increasing p53 recruitment. Moreover, luciferase reporter studies demonstrated that both κB and p53 cis-elements were required for efficient transactivation in response to temozolomide. Depletion of MALAT1 sensitized patient-derived GBM cells to temozolomide cytotoxicity, and in vivo delivery of nanoparticle-encapsulated anti-MALAT1 siRNA increased the efficacy of temozolomide in mice bearing intracranial GBM xenografts. Despite these observations, in situ hybridization of GBM specimens and analysis of publicly available datasets revealed that MALAT1 expression within GBM tissue was not prognostic of overall survival. Together, these findings support MALAT1 as a target for chemosensitization of GBM and identify p50 and p52 as primary regulators of this ncRNA. SIGNIFICANCE: These findings identify NF-κB and p53 as regulators of the lncRNA MALAT1 and suggest MALAT1 as a potential target for the chemosensitization of GBM.

Decoy Receptor DcR1 Is Induced in a p50/Bcl3-Dependent Manner and Attenuates the Efficacy of Temozolomide.

Temozolomide is used widely to treat malignant glioma, but the overall response to this agent is generally poor. Resistance to DNA-damaging drugs such as temozolomide has been related to the induction of antiapoptotic proteins. Specifically, the transcription factor NF-κB has been suggested to participate in promoting the survival of cells exposed to chemotherapy. To identify factors that modulate cytotoxicity in the setting of DNA damage, we used an unbiased strategy to examine the NF-κB-dependent expression profile induced by temozolomide. By this route, we defined the decoy receptor DcR1 as a temozolomide response gene induced by a mechanism relying upon p50/NF-κB1. A conserved NF-κB-binding sequence (κB-site) was identified in the proximal promoter and was demonstrated to be required for DcR1 induction by temozolomide. Loss-of-function and gain-of-function studies reveal that the atypical IκB protein, Bcl3, is also required for induction of DcR1 by temozolomide. Mechanistically, DcR1 attenuates temozolomide efficacy by blunting activation of the Fas receptor pathway in p53(+/+) glioma cells. Intracranial xenograft studies show that DcR1 depletion in glioma cells enhances the efficacy of temozolomide. Taken together, our results show how DcR1 upregulation mediates temozolomide resistance and provide a rationale for DcR1 targeting as a strategy to sensitize gliomas to this widely used chemotherapy.

S-phase-dependent p50/NF-кB1 phosphorylation in response to ATR and replication stress acts to maintain genomic stability.

The apical damage kinase, ATR, is activated by replication stress (RS) both in response to DNA damage and during normal S-phase. Loss of function studies indicates that ATR acts to stabilize replication forks, block cell cycle progression and promote replication restart. Although checkpoint failure and replication fork collapse can result in cell death, no direct cytotoxic pathway downstream of ATR has previously been described. Here, we show that ATR directly reduces survival by inducing phosphorylation of the p50 (NF-κB1, p105) subunit of NF-кB and moreover, that this response is necessary for genome maintenance independent of checkpoint activity. Cell free and in vivo studies demonstrate that RS induces phosphorylation of p50 in an ATR-dependent but DNA damage-independent manner that acts to modulate NF-кB activity without affecting p50/p65 nuclear translocation. This response, evident in human and murine cells, occurs not only in response to exogenous RS but also during the unperturbed S-phase. Functionally, the p50 response results in inhibition of anti-apoptotic gene expression that acts to sensitize cells to DNA strand breaks independent of damage repair. Ultimately, loss of this pathway causes genomic instability due to the accumulation of chromosomal breaks. Together, the data indicate that during S-phase ATR acts via p50 to ensure that cells with elevated levels of replication-associated DNA damage are eliminated.

Loss of Nfkb1 leads to early onset aging.

NF-κB is a major regulator of age-dependent gene expression and the p50/NF-κB1 subunit is an integral modulator of NF-κB signaling. Here, we examined Nfkb1-/- mice to investigate the relationship between this subunit and aging. Although Nfkb1-/- mice appear similar to littermates at six months of age, by 12 months they have a higher incidence of several observable age-related phenotypes. In addition, aged Nfkb1-/- animals have increased kyphosis, decreased cortical bone, increased brain GFAP staining and a decrease in overall lifespan compared to Nfkb1+/+. In vitro, serially passaged primary Nfkb1-/- MEFs have more senescent cells than comparable Nfkb1+/+ MEFs. Also, Nfkb1-/- MEFs have greater amounts of phospho-H2AX foci and lower levels of spontaneous apoptosis than Nfkb1+/+, findings that are mirrored in the brains of Nfkb1-/- animals compared to Nfkb1+/+. Finally, in wildtype animals a substantial decrease in p50 DNA binding is seen in aged tissue compared to young. Together, these data show that loss of Nfkb1 leads to early animal aging that is associated with reduced apoptosis and increased cellular senescence. Moreover, loss of p50 DNA binding is a prominent feature of aged mice relative to young. These findings support the strong link between the NF-κB pathway and mammalian aging.

DNA damage-induced cytotoxicity is mediated by the cooperative interaction of phospho-NF-κB p50 and a single nucleotide in the κB-site.

Phosphorylation of the NF-κB subunit, p50, is necessary for cytotoxicity in response to DNA methylation damage. Here, we demonstrate that serine 329 phosphorylation regulates the interaction of p50 with specific NF-κB binding elements based on the identity of a single κB-site nucleotide. Specifically, S329 phosphorylation reduces the affinity of p50 for κB-sites that have a cytosine (C) at the -1 position without affecting binding to sequences with a -1 adenine. The differential interaction between phospho-p50 and the -1 base regulates the downstream transcriptional response and underlies the inhibition of anti-apoptotic gene expression following DNA damage. In genes with multiple κB-sites, the presence of a single -1C κB-site enables inhibition of NF-κB-dependent activity. The data suggest that interaction between phospho-p50 and the -1 κB nucleotide facilitates cytotoxicity in response to DNA damage. Moreover, although conservation of the entire κB-site sequence is not seen across species, the identity of the -1 nt in critical anti-apoptotic genes is conserved such that the overall response to DNA damage is maintained.

p50 (NF-κB1) is an effector protein in the cytotoxic response to DNA methylation damage.

The functional significance of the signaling pathway induced by O(6)-methylguanine (O(6)-MeG) lesions is poorly understood. Here, we identify the p50 subunit of NF-κB as a central target in the response to O(6)-MeG and demonstrate that p50 is required for S(N)1-methylator-induced cytotoxicity. In response to S(N)1-methylation, p50 facilitates the inhibition of NF-κB-regulated antiapoptotic gene expression. Inhibition of NF-κB activity is noted to be an S phase-specific phenomenon that requires the formation of O(6)-MeG:T mismatches. Chk1 associates with p50 following S(N)1-methylation, and phosphorylation of p50 by Chk1 results in the inhibition of NF-κB DNA binding. Expression of an unphosphorylatable p50 mutant blocks inhibition of NF-κB-regulated antiapoptotic gene expression and attenuates S(N)1-methylator-induced cytotoxicity. While O(6)-MeG:T-induced, p50-dependent signaling is not sufficient to induce cell death, this pathway sensitizes cells to the cytotoxic effects of DNA breaks.