The association of the NF-B p65/p50 dimer with IB␣ plays a pivotal role in regulating its nuclear translocation and gene transcription. In addition, serine phosphorylation at various sites of the p65 subunit has been shown to be important in initiating transcription. Here we demonstrate that the regulation of nuclear translocation of p65 phosphorylated at serine 536 is not dependent on IB␣. Stimulation of either Jurkat or normal human T cells resulted in the nuclear translocation of phospho-p65 (Ser 536 ). In addition, the phospho-p65 (Ser 536 ) was not associated with either IB␣ or p50, and the nuclear translocation of phospho-p65 (Ser 536 ), but not total p65, was unaffected by the proteosome inhibitor MG-132, which blocks IB protein degradation and prevents p65/p50 dimer nuclear translocation. Accordingly, the co-expression of a dominant negative mutant of IB␣ blocked the transcriptional activity mediated by wild type but not the dominant positive p65 mutant (S536D). Furthermore, the transfection of the S536D form of p65 led to the induction of interleukin-8 transcription following stimulation, whereas the S536A form, which cannot be phosphorylated at this site, did not. Together, the findings suggest that p65 phosphorylated on serine 536 is not associated with or regulated by IB␣, that it has a distinct set of target genes, and that it may represent a noncanonical NF-B pathway that is independent of IB␣ regulation.The NF-B signaling pathway responds rapidly to a wide range of stimuli (1). Activation leads to the translocation of the transcription factors from the cytoplasm to the nucleus. The NF-B transcription factor consists of two subunits of either homo-or heterodimers of RelA/ p65, c-Rel, and p50. The complexes are held in the cytoplasm and prevented from activating transcription by a class of proteins referred to as inhibitors of NF-B or IB proteins. Upon stimulation, the IB proteins are phosphorylated by one of a number of IB kinases (IKK-␣, -␤, and -␥), ubiquitinylated, and degraded, which thereby allows the NF-B complex to translocate into the nucleus (2). However, recent findings have demonstrated the shuttling of the NF-B complex in and out of the nucleus in the absence of stimulation (3, 4). In addition to nuclear translocation of the NF-B complex, several studies have shown that the NF-B proteins are modified post-translationally, and those changes influence transcriptional activity. Examples of activation-induced posttranslational modifications include the acetylation of p65 to facilitate the retention of the NF-B complex in the nucleus (5, 6). In addition, the S-nitrosylation of cysteine 62 of p50 has been shown to affect the NF-B binding to DNA (7, 8).We have previously described the phosphorylation of various Rel proteins following the stimulation of T cells, and the phosphorylation of p50 increased the DNA binding capacity (9). Several studies have demonstrated the phosphorylation of p65 in response to various stimuli (10). Serine 276 of p65 is phosphorylated by protein kinase A during IB d...

Like in many tumor types, immunotherapy is currently under investigation to assess its potential efficacy in glioblastoma patients. Trials are under way to assess the efficacy of new immune checkpoint inhibitors including anti‐PD‐1 or CTLA4. We here investigate the expression and efficacy of a novel immune‐checkpoint inhibitor, called LAG‐3. We show that LAG‐3 is expressed in human glioblastoma samples and in a mouse glioblastoma model we show that knock out or LAG‐3 inhibition with a blocking antibody is efficacious against glioblastoma and can be used in combination with other immune checkpoint inhibitors toward complete eradication of the model glioblastoma tumors. From a mechanistic standpoint we show that LAG‐3 expression is an early marker of T cell exhaustion and therefore early treatment with LAG‐3 blocking antibody is more efficacious than later treatment. These data provide insight and support the design of trials that incorporate LAG‐3 in the treatment of glioblastoma.

The processes associated with transition to castration independent prostate cancer growth are not well understood. Cellular senescence is a stable cell cycle arrest that occurs in response to sublethal stress. It is often overcome in malignant transformation to confer a survival advantage. CCAAT/Enhancer Binding Protein (C/EBP) β function is frequently deregulated in human malignancies and interestingly, androgen dependent prostate cancer cells express primarily the LIP isoform. We found that C/EBPβ expression is negatively regulated by androgen receptor activity and that treatment of androgen dependent cell lines with anti-androgens increases C/EBPβ mRNA and protein levels. Accordingly, we also find that C/EBPβ levels are significantly elevated in primary prostate cancer samples from castration resistant compared with therapy naive patients. Chromatin immunoprecipitation demonstrated enhanced binding of the androgen receptor to the proximal promoter of the CEBPB gene in the presence of dihydroxytestosterone. Upon androgen deprivation, induction of C/EBPβ is facilitated by active transcription as evident by increased histone 3 acetylation at the C/EBPβ promoter. Also, the androgen agonist R1881 suppresses the activity of a CEBPB promoter reporter. Loss of C/EBPβ expression prevents growth arrest following androgen deprivation or anti-androgen challenge. Accordingly, suppression of C/EBPβ under low androgen conditions results in reduced expression of senescence-associated secretory genes, significantly decreased number of cells displaying heterochromatin foci, and increased numbers of Ki67 positive cells. Ectopic expression of C/EBPβ caused pronounced morphological changes, reduced PC cell growth, and increased the number of senescent LNCaP cells. Lastly, we found that senescence contributes to prostate cancer cell survival under androgen deprivation, and C/EBPβ deficient cells were significantly more susceptible to killing by cytotoxic chemotherapy following androgen deprivation. Our data demonstrate that up-regulation of C/EBPβ is critical for complete maintenance of androgen deprivation induced senescence and that targeting C/EBPβ expression may synergize with anti-androgen or chemotherapy in eradicating prostate cancer.