Faculty Bibliography

In contrast to the common role of histone deacetylases (HDACs) for gene repression, HDAC activity provides a required positive function for IFN-stimulated gene (ISG) expression. Here, we show that HDAC1/2 as components of the Sin3A complex are required for ISG transcriptional elongation but not for recruitment of RNA polymerase or transcriptional initiation. Transcriptional arrest by HDAC inhibition coincides with failure to recruit the epigenetic reader Brd4 and elongation factor P-TEFb due to sequestration of Brd4 on hyperacetylated chromatin. Brd4 availability is regulated by an equilibrium cycle between opposed acetyltransferase and deacetylase activities that maintains a steady-state pool of free Brd4 available for recruitment to inducible promoters. An ISG expression signature is a hallmark of interferonopathies and other autoimmune diseases. Combined inhibition of HDAC1/2 and Brd4 resolved the aberrant ISG expression detected in cells derived from patients with two inherited interferonopathies, ISG15 and USP18 deficiencies, defining a novel therapeutic approach to ISG-associated autoimmune diseases.

In addition to its canonical role in nuclear transcription, signal transducer and activator of transcription 3 (STAT3) is emerging as an important regulator of mitochondrial function. Here, we demonstrate that a novel inhibitor that binds with high affinity to the STAT3 SH2 domain triggers a complex cascade of events initiated by interference with mitochondrial STAT3 (mSTAT3). The mSTAT3-drug interaction leads to mitochondrial dysfunction, accumulation of proteotoxic STAT3 aggregates, and cell death. The cytotoxic effects depend directly on the drug's ability to interfere with mSTAT3 and mitochondrial function, as demonstrated by site-directed mutagenesis and use of STAT3 knockout and mitochondria-depleted cells. Importantly, the lethal consequences of mSTAT3 inhibition are enhanced by glucose starvation and by increased reliance of cancer cells and tumor-initiating cells on mitochondria, resulting in potent activity in cell cultures and tumor xenografts in mice. These findings can be exploited for eliciting synthetic lethality in metabolically stressed cancer cells using high-affinity STAT3 inhibitors. Thus, this study provides insights on the role of mSTAT3 in cancer cells and a conceptual framework for developing more effective cancer therapies.

Dendritic cells (DCs) are crucial for mediating immune responses but, when deregulated, also contribute to immunological disorders, such as autoimmunity. The molecular mechanism underlying the function of DCs is incompletely understood. In this study, we have identified TANK-binding kinase 1 (TBK1), a master innate immune kinase, as an important regulator of DC function. DC-specific deletion of Tbk1 causes T cell activation and autoimmune symptoms and also enhances antitumor immunity in animal models of cancer immunotherapy. The TBK1-deficient DCs have up-regulated expression of co-stimulatory molecules and increased T cell-priming activity. We further demonstrate that TBK1 negatively regulates the induction of a subset of genes by type I interferon receptor (IFNAR). Deletion of IFNAR1 could largely prevent aberrant T cell activation and autoimmunity in DC-conditional Tbk1 knockout mice. These findings identify a DC-specific function of TBK1 in the maintenance of immune homeostasis and tolerance.

TBLR1/TBL1XR1, a core component of the nuclear receptor corepressor (NCoR) complex critical for the regulation of multiple nuclear receptors, is a transcriptional coactivator of androgen receptor (AR) and functions as a tumor suppressor when expressed in the nucleus in prostate. Subcellular localization of a protein is critical for its function, and although TBLR1, as a transcriptional cofactor, has been primarily viewed as a nuclear protein, many cells also express variable levels of cytoplasmic TBLR1 and its cytoplasmic specific functions have not been studied. Prostate cancer (PCa) cells express moderately higher level of cytoplasmic TBLR1 compared to benign prostate cells. When comparing androgen-dependent (AD) to androgen-independent (AI) PCa, AI cells contain very high levels of TBLR1 cytoplasmic expression and low levels of nuclear expression. Overexpression of cytoplasmic TBLR1 in AD cells inhibits apoptosis induced by androgen deprivation therapy, either in an androgen free condition or in the presence of bicalutamide. Additionally, we identified a cytoplasmic specific isoform of TBLR1 (cvTBLR1) approximately 5 kDa lower in molecular weight, that is expressed at higher levels in AI PCa cells. By immunoprecipitation, we purified cvTBLR1 and using mass spectrometry analysis combined with N-terminal TMPP labeling and Edman degradation, we identified the cleavage site of cvTBLR1 at amino acid 89, truncating the first 88 amino acids of the N-terminus of the full length protein. Functionally, cvTBLR1 expressed in the cytoplasm reduced apoptosis in PCa cells and promoted growth, migration, and invasion. Finally, we identified a nuclear export signal sequence for TBLR1 cellular localization by deletion and site-directed mutagenesis. The roles of TBLR1 and cvTBLR1 provide novel insights into the mechanism of castration resistance and new strategies for PCa therapy.

To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/beta-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and beta-catenin activity. beta-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and beta-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3beta and the SWI/SNF gene Arid1b to increase beta-catenin. Knockdown of Arid1b or Gsk3beta in Stat3fl/fl;Nf1fl/fl;DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/beta-catenin pathway inhibitors in neurofibroma therapeutic trials.

Sensing invading pathogens early in infection is critical for establishing host defense. Two cytosolic RIG-like RNA helicases, RIG-I and MDA5, are key to type I interferon (IFN) induction in response to viral infection. Mounting evidence suggests that another viral RNA sensor, protein kinase R (PKR), may also be critical for IFN induction during infection, although its exact contribution and mechanism of action are not completely understood. Using PKR-deficient cells, we found that PKR was required for type I IFN induction in response to infection by vaccinia virus lacking the PKR antagonist E3L (VVDeltaE3L), but not by Sendai virus or influenza A virus lacking the IFN-antagonist NS1 (FluDeltaNS1). IFN induction required the catalytic activity of PKR, but not the phosphorylation of its principal substrate, eIF2alpha, or the resulting inhibition of host translation. In the absence of PKR, IRF3 nuclear translocation was impaired in response to MDA5 activators, VVDeltaE3L and encephalomyocarditis virus, but not during infection with a RIG-I-activating virus. Interestingly, PKR interacted with both RIG-I and MDA5; however, PKR was only required for MDA5-mediated, but not RIG-I-mediated, IFN production. Using an artificially activated form of PKR, we showed that PKR activity alone was sufficient for IFN induction. This effect required MAVS and correlated with IRF3 activation, but no longer required MDA5. Nonetheless, PKR activation during viral infection was enhanced by MDA5, as virus-stimulated catalytic activity was impaired in MDA5-null cells. Taken together, our data describe a critical and non-redundant role for PKR following MDA5, but not RIG-I, activation to mediate MAVS-dependent induction of type I IFN through a kinase-dependent mechanism.

Increased production of mitochondrial-derived reactive oxygen species (ROS) is characteristic of a metabolic shift observed during malignant transformation. While the exact sources and roles of ROS in tumorigenesis remain to be defined, it has become clear that maintaining redox balance is critical for cancer cell proliferation and survival, and as such may represent a vulnerability that can be exploited therapeutically. STAT3, a latent cytosolic transcription factor activated by diverse cytokines and growth factors, has been shown to exhibit an additional, non-transcriptional function in mitochondria, including modulation of electron transport chain activity. In particular, malignant transformation by Ras oncogenes exploits mitochondrial STAT3 functions. We used mass-spectrometry based metabolomics profiling to explore the biochemical basis for the STAT3-dependence of Ras transformation. We identified the gamma-glutamyl cycle, the production of glutathione, and the regulation of ROS as a mitochondrial-STAT3 dependent pathway in Ras-transformed cells. Experimental inhibition of key enzymes in the glutathione cycle resulted in depletion of glutathione, accumulation of ROS, oxidative DNA damage, and cell death in an oncogenic Ras and mitochondrial-STAT3 dependent manner. These data uncover a synthetic lethal interaction involving glutathione production and mitochondrial ROS regulation in Ras-transformed cells that is governed by mitochondrial STAT3 and might be exploited therapeutically.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19ARF as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14ARF expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.