Lisa M. Nilsson

DNA Damage Primes the Type I Interferon System via the Cytosolic DNA Sensor STING to Promote Anti-Microbial Innate Immunity

Dysfunction in Ataxia-telangiectasia mutated (ATM), a central component of the DNA repair machinery, results in Ataxia Telangiectasia (AT), a cancer-prone disease with a variety of inflammatory manifestations. By analyzing AT patient samples and Atm(-/-) mice, we found that unrepaired DNA lesions induce type I interferons (IFNs), resulting in enhanced anti-viral and anti-bacterial responses in Atm(-/-) mice. Priming of the type I interferon system by DNA damage involved release of DNA into the cytoplasm where it activated the cytosolic DNA sensing STING-mediated pathway, which in turn enhanced responses to innate stimuli by activating the expression of Toll-like receptors, RIG-I-like receptors, cytoplasmic DNA sensors, and their downstream signaling partners. This study provides a potential explanation for the inflammatory phenotype of AT patients and establishes damaged DNA as a cell intrinsic danger signal that primes the innate immune system for a rapid and amplified response to microbial and environmental threats.

GPS2-dependent corepressor/SUMO pathways govern anti-inflammatory actions of LRH-1 and LXRβ in the hepatic acute phase response

The orphan receptor LRH-1 and the oxysterol receptors LXRalpha and LXRbeta are established transcriptional regulators of lipid metabolism that appear to control inflammatory processes. Here, we investigate the anti-inflammatory actions of these nuclear receptors in the hepatic acute phase response (APR). We report that selective synthetic agonists induce SUMOylation-dependent recruitment of either LRH-1 or LXR to hepatic APR promoters and prevent the clearance of the N-CoR corepressor complex upon cytokine stimulation. Investigations of the APR in vivo, using LXR knockout mice, indicate that the anti-inflammatory actions of LXR agonists are triggered selectively by the LXRbeta subtype. We further find that hepatic APR responses in small ubiquitin-like modifier-1 (SUMO-1) knockout mice are increased, which is due in part to diminished LRH-1 action at APR promoters. Finally, we provide evidence that the metabolically important coregulator GPS2 functions as a hitherto unrecognized transrepression mediator of interactions between SUMOylated nuclear receptors and the N-CoR corepressor complex. Our study extends the knowledge of anti-inflammatory mechanisms and pathways directed by metabolic nuclear receptor-corepressor networks to the control of the hepatic APR, and implies alternative pharmacological strategies for the treatment of human metabolic diseases associated with inflammation.

Therapeutic Implications for the Induced Levels of Chk1 in Myc-Expressing Cancer Cells

Purpose: The transcription factor c-Myc (or “Myc”) is a master regulator of pathways driving cell growth and proliferation. MYC is deregulated in many human cancers, making its downstream target genes attractive candidates for drug development. We report the unexpected finding that B-cell lymphomas from mice and patients exhibit a striking correlation between high levels of Myc and checkpoint kinase 1 (Chk1). Experimental Design: By in vitro cell biology studies as well as preclinical studies using a genetically engineered mouse model, we evaluated the role of Chk1 in Myc-overexpressing cells. Results: We show that Myc indirectly induces Chek1 transcript and protein expression, independently of DNA damage response proteins such as ATM and p53. Importantly, we show that inhibition of Chk1, by either RNA interference or a novel highly selective small molecule inhibitor, results in caspase-dependent apoptosis that affects Myc-overexpressing cells in both in vitro and in vivo mouse models of B-cell lymphoma. Conclusion: Our data suggest that Chk1 inhibitors should be further evaluated as potential drugs against Myc-driven malignancies such as certain B-cell lymphoma/leukemia, neuroblastoma, and some breast and lung cancers. Clin Cancer Res; 17(22); 7067–79. ©2011 AACR.

BET and HDAC inhibitors induce similar genes and biological effects and synergize to kill in Myc-induced murine lymphoma

Significance Bromodomain and extraterminal (BET) proteins bind acetylated proteins, including histones, and regulate transcription. Interestingly, inhibitors of BET proteins (BETi) can block cancer cell proliferation and induce apoptosis in a wide range of tumor types. To date many of the effects of BETi have been attributed to transcriptional suppression of genes like the MYC oncogene. We show that genetically-engineered Myc-induced lymphoma mouse models are highly sensitive to BETi without MYC transcription being suppressed. Our data suggest broad effects on transcription by BETi including a set of genes being induced. Here a genetic and functional link between BET proteins and histone deacetylases is unraveled that opens up avenues for combination therapies against cancer. The bromodomain and extraterminal (BET) domain family of proteins binds to acetylated lysines on histones and regulates gene transcription. Recently, BET inhibitors (BETi) have been developed that show promise as potent anticancer drugs against various solid and hematological malignancies. Here we show that the structurally novel and orally bioavailable BET inhibitor RVX2135 inhibits proliferation and induces apoptosis of lymphoma cells arising in Myc-transgenic mice in vitro and in vivo. We find that BET inhibition exhibits broad transcriptional effects in Myc-transgenic lymphoma cells affecting many transcription factor networks. By examining the genes induced by BETi, which have largely been ignored to date, we discovered that these were similar to those induced by histone deacetylase inhibitors (HDACi). HDACi also induced cell-cycle arrest and cell death of Myc-induced murine lymphoma cells and synergized with BETi. Our data suggest that BETi sensitize Myc-overexpressing lymphoma cells partly by inducing HDAC-silenced genes, and suggest synergistic and therapeutic combinations by targeting the genetic link between BETi and HDACi.

Myc targets Cks1 to provoke the suppression of p27Kip1, proliferation and lymphomagenesis

Reduced levels of the cyclin‐dependent kinase inhibitor p27Kip1 connote poor prognosis in cancer. In human Burkitt lymphoma and in precancerous B cells and lymphomas arising in Eμ‐Myc transgenic mice, p27Kip1 expression is markedly reduced. We show that the transcription of the Cks1 component of the SCFSkp2 complex that is necessary for p27Kip1 ubiquitylation and degradation is induced by Myc. Further, Cks1 expression is elevated in precancerous Eμ‐Myc B cells, and high levels of Cks1 are also a hallmark of Eμ‐Myc lymphoma and of human Burkitt lymphoma. Finally, loss of Cks1 in Eμ‐Myc B cells elevates p27Kip1 levels, reduces proliferation and markedly delays lymphoma development and dissemination of disease. Therefore, Myc suppresses p27Kip1 expression, accelerates cell proliferation and promotes tumorigenesis at least in part through its ability to selectively induce Cks1.

Involvement of corepressor complex subunit GPS2 in transcriptional pathways governing human bile acid biosynthesis.

Coordinated regulation of bile acid biosynthesis, the predominant pathway for hepatic cholesterol catabolism, is mediated by few key nuclear receptors including the orphan receptors liver receptor homolog 1 (LRH-1), hepatocyte nuclear factor 4α (HNF4α), small heterodimer partner (SHP), and the bile acid receptor FXR (farnesoid X receptor). Activation of FXR initiates a feedback regulatory loop via induction of SHP, which suppresses LRH-1- and HNF4α-dependent expression of cholesterol 7α hydroxylase (CYP7A1) and sterol 12α hydroxylase (CYP8B1), the two major pathway enzymes. Here we dissect the transcriptional network governing bile acid biosynthesis in human liver by identifying GPS2, a stoichiometric subunit of a conserved corepressor complex, as a differential coregulator of CYP7A1 and CYP8B1 expression. Direct interactions of GPS2 with SHP, LRH-1, HNF4α, and FXR indicate alternative coregulator recruitment strategies to cause differential transcriptional outcomes. In addition, species-specific differences in the regulation of bile acid biosynthesis were uncovered by identifying human CYP8B1 as a direct FXR target gene, which has implications for therapeutic approaches in bile acid-related human disorders.

Mouse Genetics Suggests Cell-Context Dependency for Myc-Regulated Metabolic Enzymes during Tumorigenesis

c-Myc (hereafter called Myc) belongs to a family of transcription factors that regulates cell growth, cell proliferation, and differentiation. Myc initiates the transcription of a large cast of genes involved in cell growth by stimulating metabolism and protein synthesis. Some of these, like those involved in glycolysis, may be part of the Warburg effect, which is defined as increased glucose uptake and lactate production in the presence of adequate oxygen supply. In this study, we have taken a mouse-genetics approach to challenge the role of select Myc-regulated metabolic enzymes in tumorigenesis in vivo. By breeding λ-Myc transgenic mice, Apc Min mice, and p53 knockout mice with mouse models carrying inactivating alleles of Lactate dehydrogenase A (Ldha), 3-Phosphoglycerate dehydrogenase (Phgdh) and Serine hydroxymethyltransferase 1 (Shmt1), we obtained offspring that were monitored for tumor development. Very surprisingly, we found that these genes are dispensable for tumorigenesis in these genetic settings. However, experiments in fibroblasts and colon carcinoma cells expressing oncogenic Ras show that these cells are sensitive to Ldha knockdown. Our genetic models reveal cell context dependency and a remarkable ability of tumor cells to adapt to alterations in critical metabolic pathways. Thus, to achieve clinical success, it will be of importance to correctly stratify patients and to find synthetic lethal combinations of inhibitors targeting metabolic enzymes.

Physiological Differences between Human and Rat Primary Hepatocytes in Response to Liver X Receptor Activation by 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic Acid Hydrochloride (GW3965)

The liver is central to the maintenance of glucose and lipid homeostasis, and liver X receptors (LXRs) are key regulators of expression of the genes involved. So far, effects of activation of LXR in human hepatocytes have not been well characterized. Here we show that treatment of primary human hepatocytes with the synthetic LXR ligand 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic acid hydrochloride (GW3965) results in reduced output of bile acids and very low density lipoprotein triglycerides and induced expression of adipose differentiation-related protein accompanied by increased lipid storage. Genome wide-expression profiling identified novel human LXR target genes in the glycolytic and lipogenic pathways and indicated that LXR activation reduced hepatic insulin sensitivity. Comparative experiments showed significant differences in the response to GW3965 between human and rat hepatocytes, raising the question as to how well rodent models reflect the human situation. In summary, the risk of hepatic steatosis upon pharmaceutical targeting of LXR may be a particularly serious consequence in humans.

Skp2 directs Myc-mediated suppression of p27Kip1 yet has modest effects on Myc-driven lymphomagenesis

The universal cyclin-dependent kinase inhibitor p27Kip1 functions as a tumor suppressor, and reduced levels of p27Kip1 connote poor prognosis in several human malignancies. p27Kip1 levels are predominately regulated by ubiquitin-mediated turnover of the protein, which is marked for destruction by the E3 ubiquitin ligase SCFSkp2 complex following its phosphorylation by the cyclin E–cyclin-dependent kinase 2 complex. Binding of phospho-p27Kip1 is directed by the Skp2 F-box protein, and this is greatly augmented by its allosteric regulator Cks1. We have established that programmed expression of c-Myc in the B cells of Eμ-Myc transgenic mice triggers p27Kip1 destruction by inducing Cks1, that this response controls Myc-driven proliferation, and that loss of Cks1 markedly delays Myc-induced lymphomagenesis and cancels the dissemination of these tumors. Here, we report that elevated levels of Skp2 are a characteristic of Eμ-Myc lymphomas and of human Burkitt lymphoma that bear MYC/Immunoglobulin chromosomal translocations. As expected, Myc-mediated suppression of p27Kip1 was abolished in Skp2-null Eμ-Myc B cells. However, the effect of Skp2 loss on Myc-driven proliferation and lymphomagenesis was surprisingly modest compared with the effects of Cks1 loss. Collectively, these findings suggest that Cks1 targets, in addition to p27Kip1, are critical for Myc-driven proliferation and tumorigenesis. Mol Cancer Res; 8(3); 353–62

Id2 is dispensable for Myc-induced lymphomagenesis

The Eμ-Myc transgenic mouse appears to be an accurate model of human Burkitt’s lymphoma that bears MYC/Immunoglobulin gene translocations. Id2, a negative regulator of basic helix-loop-helix transcription factors, has also been proposed as a Myc target gene that drives the proliferative response of Myc by binding to and overriding the checkpoint functions of the retinoblastoma tumor suppressor protein. Targeted deletion of Id2 in mice results in defects in B-cell development and prevents the development of peripheral lymphoid nodes. In precancerous B cells and lymphomas that arise in Eμ-Myc transgenic mice and in Burkitt’s lymphomas, Id2 is overexpressed, suggesting that it plays a regulatory role in lymphoma development. Surprisingly, despite these connections, Eμ-Myc mice lacking Id2 succumb to lethal B-cell lymphoma at rates comparable with wild-type Eμ-Myc transgenics. Furthermore, precancerous splenic B cells lacking Id2 do not exhibit any significant defects in Myc-induced target gene transactivation and proliferation. However, due to their lack of secondary lymph nodes, Eμ-Myc mice lacking Id2 rather succumb to disseminated lymphoma with an associated leukemia, with pronounced infiltrates of the bone marrow and other major organs. Collectively these findings argue that targeting Id2 functions may be ineffective in preventing Myc-associated malignancies.