PBRM1 bromodomains associate with RNA to facilitate chromatin association.

PBRM1 is a subunit of the PBAF chromatin remodeling complex, which is mutated in 40-50% of clear cell renal cell carcinoma patients. It is thought to largely function as a chromatin binding subunit of the PBAF complex, but the molecular mechanism underlying this activity is not fully known. PBRM1 contains six tandem bromodomains which are known to cooperate in binding of nucleosomes acetylated at histone H3 lysine 14 (H3K14ac). Here, we demonstrate that the second and fourth bromodomains from PBRM1 also bind nucleic acids, selectively associating with double stranded RNA elements. Disruption of the RNA binding pocket is found to compromise PBRM1 chromatin binding and inhibit PBRM1-mediated cellular growth effects.

Identification of novel nuclear targets of human thioredoxin 1.

The dysregulation of protein oxidative post-translational modifications has been implicated in stress-related diseases. Trx1 is a key reductase that reduces specific disulfide bonds and other cysteine post-translational modifications. Although commonly in the cytoplasm, Trx1 can also modulate transcription in the nucleus. However, few Trx1 nuclear targets have been identified because of the low Trx1 abundance in the nucleus. Here, we report the large-scale proteomics identification of nuclear Trx1 targets in human neuroblastoma cells using an affinity capture strategy wherein a Trx1C35S mutant is expressed. The wild-type Trx1 contains a conserved C32XXC35 motif, and the C32 thiol initiates the reduction of a target disulfide bond by forming an intermolecular disulfide with one of the oxidized target cysteines, resulting in a transient Trx1-target protein complex. The reduction is rapidly consummated by the donation of a C35 proton to the target molecule, forming a Trx1 C32-C35 disulfide, and results in the concurrent release of the target protein containing reduced thiols. By introducing a point mutation (C35 to S35) in Trx1, we ablated the rapid dissociation of Trx1 from its reduction targets, thereby allowing the identification of 45 putative nuclear Trx1 targets. Unexpectedly, we found that PSIP1, also known as LEDGF, was sensitive to both oxidation and Trx1 reduction at Cys 204. LEDGF is a transcription activator that is vital for regulating cell survival during HIV-1 infection. Overall, this study suggests that Trx1 may play a broader role than previously believed that might include regulating transcription, RNA processing, and nuclear pore function in human cells.

Thioredoxin 1-mediated post-translational modifications: reduction, transnitrosylation, denitrosylation, and related proteomics methodologies.

Despite the significance of redox post-translational modifications (PTMs) in regulating diverse signal transduction pathways, the enzymatic systems that catalyze reversible and specific oxidative or reductive modifications have yet to be firmly established. Thioredoxin 1 (Trx1) is a conserved antioxidant protein that is well known for its disulfide reductase activity. Interestingly, Trx1 is also able to transnitrosylate or denitrosylate (defined as processes to transfer or remove a nitric oxide entity to/from substrates) specific proteins. An intricate redox regulatory mechanism has recently been uncovered that accounts for the ability of Trx1 to catalyze these different redox PTMs. In this review, we will summarize the available evidence in support of Trx1 as a specific disulfide reductase, and denitrosylation and transnitrosylation agent, as well as the biological significance of the diverse array of Trx1-regulated pathways and processes under different physiological contexts. The dramatic progress in redox proteomics techniques has enabled the identification of an increasing number of proteins, including peroxiredoxin 1, whose disulfide bond formation and nitrosylation status are regulated by Trx1. This review will also summarize the advancements of redox proteomics techniques for the identification of the protein targets of Trx1-mediated PTMs. Collectively, these studies have shed light on the mechanisms that regulate Trx1-mediated reduction, transnitrosylation, and denitrosylation of specific target proteins, solidifying the role of Trx1 as a master regulator of redox signal transduction.

New and old functions of STAT3: a pivotal target for individualized treatment of cancer.

Signal transducers and activators of transcription (STAT) regulate a plethora of cytokine responses. Recently, aberrant signaling by STAT proteins has been demonstrated to play important roles in the pathogenesis of many neoplasms, by promoting cell cycle progression and survival, stimulating angiogenesis, and impairing immunological responses and tumor surveillance. We have developed genetic tools to evaluate STAT-dependent malignancy and showed that survival and growth of lymphoid malignancies requires expression of STAT3. In contrast, loss of STAT3 in normal cells does not impair their growth or survival; but in spite of this apparent dispensability of STAT3, STAT3-null fibroblasts are resistant to transformation by a variety of oncogenes. The precise molecular mechanisms responsible for the tumorigenic activity of STAT3 have been only partially elucidated. While the tyrosine phosphorylation of STAT3, which is indicative of its signal-dependent activation, is a common occurrence in tumors, and appears to play a crucial role in some malignancies, a variety of new data suggest that it can be dispensable under some circumstances and STAT3 can participate in transformation through novel and non-canonical mechanisms. The discovery and dissection of non-canonical modes of STAT3 action will open new avenues for the design of effective therapeutics capable of neutralizing the tumorigenic properties of this molecule.

Stat3 is required for ALK-mediated lymphomagenesis and provides a possible therapeutic target.

Anaplastic large cell lymphomas (ALCLs) are caused by chromosomal translocations that juxtapose the anaplastic lymphoma kinase (ALK) proto-oncogene to a dimerization partner, resulting in constitutive expression of ALK and ALK tyrosine kinase activity. One substrate of activated ALK in human ALCLs is the transcription factor Stat3, and its phosphorylation is accurately recapitulated in a new nucleophosmin (NPM)-ALK transgenic mouse model of lymphomagenesis. Here we show by gene targeting that Stat3 is required for the transformation of mouse embryonic fibroblasts in vitro, for the development of B-cell lymphoma in transgenic mice and for the growth and survival of both human and mouse NPM-ALK-transformed B and T cells. Ablation of Stat3 expression by antisense oligonucleotides significantly (P < 0.0001) impaired the growth of human and mouse NPM-ALK tumors in vivo. Pharmacological ablation of Stat3 represents a new candidate approach for the treatment of human lymphoma

Tim-3+ T-bet+ tumor-specific Th1 cells colocalize with and inhibit development and growth of murine neoplasms.

Although T cells infiltrate many types of murine and human neoplasms, in many instances tumor-specific cytotoxicity is not observed. Strategies to stimulate CTL-mediated antitumor immunity have included in vitro stimulation and/or genetic engineering of T cells, followed by adoptive transfer into tumor-bearing hosts. In this model of B cell lymphoma in SJL/J mice, we used Tim-3(+) T-bet(+) Th1 cells to facilitate the development of tumor-specific CTL. Tumor-specific Th1 cell lines were polarized with IL-12 during in vitro stimulation and long term maintenance. As few as 5 million Tim-3(+) T-bet(+) Th1 cells enabled recipients to resist growth of malignant transplantable cells. In addition, similar numbers of Th1 cells injected into 2- to 3-mo-old mice inhibited development of the spontaneous primary lymphomas, which normally arise in 90% of aging mice. CFSE(+) Th1 cells colocalized with injected tumor cells in vivo and formed conjugates with the tumor cells within follicles, whereas in nontumor-challenged recipients the CFSE(+) Th1 cells localized only within the T cell zones of the spleen. These results provide evidence that adoptive immunotherapy with Tim-3(+) T-bet(+) tumor-specific Th1 cells can be used to induce host cytotoxic responses that inhibit the development and growth of neoplastic cells.

NPM-ALK transgenic mice spontaneously develop T-cell lymphomas and plasma cell tumors.

Anaplastic Large Cell Lymphomas (ALCLs) carry translocations in which the anaplastic lymphoma kinase (ALK) gene is juxtaposed to various genes, the most common of which is the NPM/B23 gene. ALK fusion proteins result in the constitutive activation of ALK tyrosine kinase, thereby enhancing proliferation and increasing cell survival. A direct role for NPM-ALK in cellular transformation has been shown in vitro with immortalized cell lines and in vivo using retroviral transfer experiments. Nonetheless, there is no direct evidence of its oncogenic potential in T lymphocytes, which represent the most common target of ALK chimeras. Here, we describe a new mouse model of lymphomagenesis in which human NPM-ALK transcription was targeted to T cells. NPM-ALK transgenic (Tg) mice were born with the expected mendelian distribution, normal lymphoid organs, and a normal number and proportion of helper and suppressor T cells. However, after a short period of latency, all NPM-ALK Tg mice developed malignant lymphoproliferative disorders (mean survival, 18 weeks). NPM-ALK Tg thymic lymphomas displayed a T-cell phenotype characteristic of immature thymocytes and frequently coexpressed surface CD30. A subset of the NPM-ALK Tg mice also developed clonal B-cell plasma cell neoplasms. These tumors arose in peripheral lymphoid organs (plasmacytomas) or within the bone marrow and often led to peripheral neuropathies and limb paralysis. Our NPM-ALK Tg mice are a suitable model to dissect the molecular mechanisms of ALK-mediated transformation and to investigate the efficacy of new therapeutic approaches for the treatment of human ALCL in vivo.

Regulation of mouse mammary tumor virus env transcriptional activator initiated mammary tumor virus superantigen transcripts in lymphomas of SJL/J mice: role of Ikaros, demethylation, and chromatin structural change in the transcriptional activation of mammary tumor virus superantigen.

Mammary tumor virus (Mtv29)-encoded superantigen expressed by SJL/J mouse B cell lymphomas stimulates CD4+V16+ T cells and thereby acquires T cell help necessary for lymphoma growth. Mtv29 mouse mammary tumor virus env transcriptional activator (META) env-controlled Mtv29 superantigen (vSAg29) mRNA transcripts (1.8 kb) are not expressed in normal B or other somatic cells. Real-time PCR-based assays with DNA from normal SJL liver and vSAg29- lymphoma (cNJ101), digested with methylation-sensitive enzymes, showed hypermethylation at AvaI, FspI, HpaII, ThaI, and the distal HgaI sites of the META env, but vSAg29+ lymphoma cells showed significant demethylation at AvaI, HpaII, and the distal HgaI sites. The distal HgaI site that is adjacent to an Ikaros binding site is significantly demethylated in the META env DNA from primary lymphomas. Gel shift assays showed binding of Ikaros to a sequence representing this region in the META env. SJL lymphomas expressed the Ikaros isoform Ik6 that was absent in normal B cells. vSAg29+ cells exhibited increased DNaseI accessibility to chromatin at the vSAg29 initiation site. Treatment of cNJ101 cells with a demethylating agent, 5-azacytidine, and a histone deacetylase inhibitor, trichostatin A, caused hypomethylation at AvaI, HpaII, and distal HgaI sites and led to chromatin structural change at the vSAg29 initiation site, accompanied by the expression of vSAg29 transcripts. This enabled cNJ101 cells to stimulate SJL lymphoma-responsive CD4+V16+ T hybridoma cells. Thus, demethylation at the distal HgaI site of the Mtv29 META env permits vSAg29 expression, which may have an impact on the development of germinal center-derived B cell lymphomas of SJL/J mice.

Interleukin-12-induced cytotoxicity against syngeneic B cell lymphomas of SJL/J mice.

The B cell lymphomas (RCS) that develop spontaneously in 90% of aging SJL/J mice stimulate syngeneic CD4+ Vbeta16+ Th2 cells to produce cytokines, such as IL-4 and IL-5, which promote lymphoma growth. Although RCS cells express a unique superantigen (vSAg) encoded by an endogenous MMTV (Mtv-29) provirus that also elicits IFN-gamma production from naïve syngeneic lymphoid cells, there is no development of RCS-specific cytotoxicity. However, addition of IL-12 to co-cultures of SJL spleen and irradiated (gamma-)RCS cells resulted in the appearance of effector cells that killed RCS and NK-susceptible target cells. Antibody depletion studies revealed at least two types of RCS/IL-12-induced cytotoxic cells: (1) NK cells (Asialo GM1+) and (2) CD8+ CTL. Despite high titers of IFN-gamma in the SN of co-culture of SJL spleen and gamma-RCS cells, cytotoxicity only developed if IL-12 was also included in the co-cultures. The results of RNAse protection assays and multi-parameter FACS analysis demonstrated an upregulation of IFN-gamma and decrease in IL-4 by activated Th cells in co-cultures with IL-12. These results indicate that inclusion of IL-12 in primary co-cultures of SJL spleen and gamma-RCS cells influences the qualitative nature of the response to favor use of RCS-responsive Th1 rather than Th2 cells to facilitate the production of cytotoxic effector cells. Results of in vivo experiments support this hypothesis, as judged by tumor growth assays and FACS analysis of the tumor cell content of lymphoid tissues. Inhibition of lymphoma growth was observed in mice given gamma-RCS/IL-12-induced effector cells prior to injection of viable RCS cells. These results demonstrate that IL-12 can be used to alter the host immune response leading to induction of cytotoxic effector cells that inhibit the development and/or progressive growth of otherwise resistant B cell lymphomas in SJL/J mice.